U.S. patent application number 16/481617 was filed with the patent office on 2019-12-05 for cardiac progenitor cells having enhanced p53 expression and uses thereof.
This patent application is currently assigned to AAL SCIENTIFICS, INC.. The applicant listed for this patent is AAL SCIENTIFICS, INC.. Invention is credited to Piero ANVERSA, Annarosa LERI.
Application Number | 20190365822 16/481617 |
Document ID | / |
Family ID | 63041079 |
Filed Date | 2019-12-05 |
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United States Patent
Application |
20190365822 |
Kind Code |
A1 |
ANVERSA; Piero ; et
al. |
December 5, 2019 |
CARDIAC PROGENITOR CELLS HAVING ENHANCED p53 EXPRESSION AND USES
THEREOF
Abstract
Disclosed herein are compositions comprising cardiac progenitor
cells that express exogenous p53 protein. Such compositions are
useful for treating cardiac diseases or disorders. Also disclosed
herein are methods of producing cardiac progenitor cells that
express exogenous p53.
Inventors: |
ANVERSA; Piero; (New York,
NY) ; LERI; Annarosa; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AAL SCIENTIFICS, INC. |
New York |
NY |
US |
|
|
Assignee: |
AAL SCIENTIFICS, INC.
New York
NY
|
Family ID: |
63041079 |
Appl. No.: |
16/481617 |
Filed: |
February 1, 2018 |
PCT Filed: |
February 1, 2018 |
PCT NO: |
PCT/US2018/016372 |
371 Date: |
July 29, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62453421 |
Feb 1, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/00 20180101; A61K
35/34 20130101; C12N 5/0657 20130101 |
International
Class: |
A61K 35/34 20060101
A61K035/34; C12N 5/077 20060101 C12N005/077; A61P 9/00 20060101
A61P009/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under Grant
No. NIA/R01AG37490 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A method of treating or preventing a heart disease or disorder
in a subject in need thereof comprising administering isolated
cardiac progenitor cells (CPCs) to the subject, wherein the CPCs
comprise one or more copies of a tumor suppressor p53 gene in
addition to the endogenous copy of a p53 gene.
2. The method of claim 1, wherein the heart disease or disorder is
heart failure, diabetic heart disease, rheumatic heart disease,
hypertensive heart disease, ischemic heart disease, cerebrovascular
heart disease, inflammatory heart disease and/or congenital heart
disease.
3. The method of claim 1, wherein the CPCs express an increased
amount of p53 protein compared to the amount expressed by CPCs that
do not comprise one or more copies of a p53 gene in addition to the
endogenous copy of a p53 gene.
4. A method of repairing and/or regenerating damaged tissue of a
heart in a subject in need thereof comprising: (a) extracting
cardiac progenitor cells (CPCs) from a heart; (b) introducing one
or more tumor suppressor p53 genes into the CPCs of step (a); (c)
culturing and expanding said CPCs from step (b); and (d)
administering a dose of said CPCs from step (c) to an area of
damaged tissue in the subject.
5. The method of claim 4, wherein the dose of said CPCs
administered to the area of damaged tissue in the subject is
effective to (i) repair and/or regenerate the damaged tissue of the
heart, and/or (ii) to promote cellular engraftment and growth of
the CPCs in the damaged tissue of the heart in a subject in need
thereof.
6. The method of claim 4, wherein the subject has diabetes.
7. A method of producing a large quantity of cardiac progenitor
cells (CPCs) comprising: (a) isolating CPCs from heart tissue; (b)
introducing one or more tumor suppressor p53 genes into the CPCs of
step (a); and (c) culturing and expanding the CPCs of step (b),
thereby (i) producing a large quantity of CPCs, (ii) producing CPCs
having an improved ability to tolerate oxidative stress compared to
CPC's from step (a), (iii) producing CPCs having restored DNA
integrity compared to CPCs from step (a), and/or (iv) producing
CPCs having an improved proliferative capacity compared to CPCs
from step (a).
8. A method of promoting cellular engraftment and growth of cells
in an organ or tissue during cell therapy, comprising: (a)
extracting cells from an organ or tissue; (b) introducing one or
more tumor suppressor p53 genes into the cells of step (a); (c)
culturing and expanding said cells from step (b); and (d) applying
an amount of said cells from step (c) to an area of damaged organ
or tissue, thereby promoting cellular engraftment and growth of
cells in the damaged organ or tissue.
9. The method of claim 7, wherein culturing and expanding the CPCs
of step (b) thereby produces CPCs having an improved ability to
tolerate oxidative stress compared to CPCs from step (a).
10. The method of claim 7, wherein culturing and expanding the CPCs
of step (b) thereby produces CPCs having restored DNA integrity
compared to CPCs from step (a).
11. The method of claim 7, wherein culturing and expanding the CPCs
of step (b) thereby produces CPCs having an improved proliferative
capacity compared to CPCs from step (a).
12. A pharmaceutical composition comprising a therapeutically
effective amount of isolated cardiac progenitor cells (CPCs) and a
pharmaceutically acceptable carrier, wherein said isolated CPCs
comprise one or more copies of a tumor suppressor p53 gene in
addition to the endogenous copy of a p53 gene.
13. The pharmaceutical composition of claim 12, wherein the
pharmaceutically acceptable carrier is for (i) repairing and/or
regenerating damaged tissue of a heart, or (ii) promoting cellular
engraftment and growth of the CPCs in damaged tissue of a
heart.
14. A pharmaceutical composition comprising a therapeutically
effective amount of cells and a pharmaceutically acceptable carrier
for promoting cellular engraftment and growth of the cells in a
damaged organ or tissue, wherein said cells comprise one or more
copies of a tumor suppressor p53 gene in addition to the endogenous
copy of a p53 gene.
15. The method of claim 5, wherein the subject has diabetes.
16. The method of claim 7, wherein culturing and expanding the CPCs
of step (b) thereby produces a large quantity of CPCs.
Description
[0001] This application claims priority to and benefit of U.S.
Provisional Patent Application No. 62/453,421, filed on Feb. 1,
2017. The contents of this application are herein incorporated by
reference in their entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0003] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
AALS_007_01 WO_SeqList_ST25.txt; date recorded: Feb. 1, 2018; file
size 3,745 bytes).
FIELD OF THE INVENTION
[0004] The present invention relates generally to the field of
cardiology. More specifically, the invention relates to cardiac
progenitor cells that express exogenous p53 protein and the use of
such cells to treat or prevent heart diseases or disorders.
BACKGROUND OF THE INVENTION
[0005] Myocardial aging in animals and humans is characterized by
an increase in number of resident cardiac progenitor cells (CPCs)
expressing the senescence-associated protein p16.sup.INK4a, which
prevents permanently the reentry of stem cells into the cell cycle
(Beausejour and Campisi, 2006, Dimmeler and Leri, 2008, Sanada et
al., 2014, Leri et al., 2015). This age-dependent effect results in
a reduction of the pool of functionally-competent CPCs in the old
heart (Torella et al., 2004). Alterations of coronary blood flow
and defects in the structural determinants of tissue oxygenation in
the aging myocardium (Hachamovitch et al., 1989) create hypoxic
micro-domains where CPCs are maintained in a quiescent state
(Sanada et al., 2014), impairing the activation of a compartment of
progenitor cells with relatively intact replicative reserve.
[0006] Ongoing clinical trials with autologous cardiac stem cells
(CSCs) are faced with a critical limitation which is related to the
modest amount of retained cells within the damaged myocardium.
There is a need for compositions and methods that can be used to
restore the structural and functional integrity of the
decompensated heart.
SUMMARY OF THE INVENTION
[0007] In one embodiment, provided herein is a method of treating
or preventing a heart disease or disorder in a subject in need
thereof comprising administering isolated cardiac progenitor cells
(CPCs) to the subject, wherein the CPCs comprise one or more copies
of a tumor suppressor p53 gene in addition to the endogenous copy
of a p53 gene. In some embodiments, the heart disease or disorder
is heart failure, diabetic heart disease, rheumatic heart disease,
hypertensive heart disease, ischemic heart disease, cerebrovascular
heart disease, inflammatory heart disease and/or congenital heart
disease. In some embodiments, the CPCs express an increased amount
of p53 protein compared to the amount expressed by CPCs that do not
comprise one or more copies of a p53 gene in addition to the
endogenous copy of a p53 gene.
[0008] In one embodiment, the invention provides a method of
repairing and/or regenerating damaged tissue of a heart in a
subject in need thereof comprising: (a) extracting cardiac
progenitor cells (CPCs) from a heart; (b) introducing one or more
tumor suppressor p53 genes into the CPCs of step (a); (c) culturing
and expanding said CPCs from step (b); and (d) administering a dose
of said CPCs from step (c) to an area of damaged tissue in the
subject effective to repair and/or regenerate the damaged tissue of
the heart. In some cases, the subject has diabetes.
[0009] In one embodiment, the invention provides a method of
promoting cellular engraftment and growth of cardiac progenitor
cells (CPCs) in damaged tissue of a heart in a subject in need
thereof comprising: (a) extracting cardiac progenitor cells (CPCs)
from a heart; (b) introducing one or more tumor suppressor p53
genes into the CPCs of step (a); (c) culturing and expanding said
CPCs from step (b); and (d) administering a dose of said CPCs from
step (c) to an area of damaged tissue in the subject effective to
promote cellular engraftment and growth of the CPCs in the damaged
tissue of the heart in a subject in need thereof. In some cases,
the subject has diabetes.
[0010] The invention further provides a method of producing a large
quantity of cardiac progenitor cells (CPCs) comprising: (a)
isolating CPCs from heart tissue: (b) introducing one or more tumor
suppressor p53 genes into the CPCs of step (a); and (c) culturing
and expanding the CPCs of step (b), thereby producing a large
quantity of CPCs.
[0011] In one embodiment, the invention provides a method of
promoting cellular engraftment and growth of cells in an organ or
tissue during cell therapy, comprising: (a) extracting cells from
an organ or tissue; (b) introducing one or more tumor suppressor
p53 genes into the cells of step (a); (c) culturing and expanding
said cells from step (b); and (d) applying an amount of said cells
from step (c) to an area of damaged organ or tissue, thereby
promoting cellular engraftment and growth of cells in the damaged
organ or tissue.
[0012] In one embodiment, the invention provides a method of
producing isolated cardiac progenitor cells (CPCs) having an
improved ability to tolerate oxidative stress, comprising: (a)
isolating CPCs from heart tissue; (b) introducing one or more tumor
suppressor p53 genes into the CPCs of step (a); and (c) culturing
and expanding the CPCs of step (b), thereby producing CPCs having
an improved ability to tolerate oxidative stress compared to CPCs
from step (a).
[0013] In one embodiment, the invention provides a method of
producing isolated cardiac progenitor cells (CPCs) having restored
DNA integrity, comprising: (a) isolating CPCs from heart tissue;
(b) introducing one or more tumor suppressor p53 genes into the
CPCs of step (a); and (c) culturing and expanding the CPCs of step
(b), thereby producing CPCs having restored DNA integrity compared
to CPCs from step (a).
[0014] In one embodiment, the invention provides a method of
producing isolated cardiac progenitor cells (CPCs) having an
improved proliferative capacity, comprising: (a) isolating CPCs
from heart tissue; (b) introducing one or more tumor suppressor p53
genes into the CPCs of step (a); and (c) culturing and expanding
the CPCs of step (b), thereby producing CPCs having an improved
proliferative capacity compared to CPCs from step (a).
[0015] In one embodiment, the invention provides a pharmaceutical
composition comprising a therapeutically effective amount of
isolated cardiac progenitor cells (CPCs) and a pharmaceutically
acceptable carrier for repairing and/or regenerating damaged tissue
of a heart, wherein said isolated CPCs comprise one or more copies
of a tumor suppressor p53 gene in addition to the endogenous copy
of a p53 gene.
[0016] In another embodiment, the invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of isolated cardiac progenitor cells (CPCs) and a
pharmaceutically acceptable carrier for promoting cellular
engraftment and growth of the CPCs in damaged tissue of a heart,
wherein said isolated CPCs comprise one or more copies of a tumor
suppressor p53 gene in addition to the endogenous copy of a p53
gene.
[0017] In one embodiment, the invention provides a pharmaceutical
composition comprising a therapeutically effective amount of cells
and a pharmaceutically acceptable carrier for promoting cellular
engraftment and growth of the cells in a damaged organ or tissue,
wherein said cells comprise one or more copies of a tumor
suppressor p53 gene in addition to the endogenous copy of a p53
gene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1C depict results showing that aging and p53 do not
alter cardiac and myocyte function. (FIG. 1A) Hemodynamics in
young-adult (3-6 months) and old (24-31 months) p53-tg and WT mice
(young WT, n=9, young p53-tg, n=7; old WT, n=11, old p53-tg, n=6).
LV SP, LV systolic pressure; LV EndDP, LV end-diastolic pressure;
LV DevP, LV developed pressure. (FIG. 1B) Ca.sup.2+ transients and
sarcomere shortening of cardiomyocytes in young WT (n=112 cells
from 10 mice) and young p53-tg (n=79 cells from .+-.7 mice). (FIG.
1C) Ca.sup.2+ transients and sarcomere shortening of cardiomyocytes
in old WT (n=40 cells from 3 mice) and old p53-tg (n=25 cells from
3 mice). "LV" refers to "left ventricle".
[0019] FIGS. 2A-2I depict characterization of p53, cardiomyocytes
and CPCs in WT and p53-tg mice. (FIGS. 2A-2B) Ki67-positive (FIG.
2A) and apoptotic TUNEL-positive (FIG. 2B) cardiomyocytes in
young-adult, 8-11 months (WT: n=9; p53-tg: n=7), and old, 20-25
months (WT: n=6; p53-tg: n=8), WT and p53-tg mice. *p<0.05 vs.
young-adult WT; **p<0.05 vs. old WT; ***p<0.05 vs.
young-adult p53-tg. (FIG. 2C) p16.sup.INK4a-positive cardiomyocytes
in old, 18-33 months, WT (n=4) and p53-tg (n=9) mice. (FIGS. 2D-2E)
Number of c-kit-positive CPCs in atrial myocardium (FIG. 2D) and
fraction of cycling Ki67-positive CPCs (FIG. 2E). WT: n=3; p53-tg:
n=4. (FIG. 2F) Population doubling time (PDT) in WT-CPCs (WT; n=3)
and p53-tg-CPCs (p53-tg; n=3). (FIG. 2G) Fraction of Ki67 labeled
WT-CPCs (n=3) and p53-tg-CPCs (n=3). (FIG. 2H) Fraction of
p16.sup.INK4a labeled WT-CPCs (n=3) and p53-tg-CPCs (n=3). (FIG.
2I) Apoptosis of WT-CPCs (n=3) and p53-tg-CPCs (n=3) measured by
Annexin V assay. In all cases data are shown as mean.+-.SD.
*p<0.05 vs. WT.
[0020] FIGS. 3A-3F show that p53 improves the DDR of CPCs. (a)
Nuclei from p53-tg-CPCs in the absence (Control) and in the
presence of doxorubicin (Doxo) are stained by DAPI (blue, left
panels); immunolabeled .gamma.H2A.X is shown in these nuclei
(green, right panels). Scale bar: 100 .mu.m. (b) Fraction of
.gamma.H2A.X-positive CPCs in the absence (control, Ctrl) and
following exposure to Doxo (Doxo): Ctrl WT-CPCs (4284 cells from 3
mice); Ctrl p53-tg-CPCs (13,334 cells from 3 mice); Doxo WT-CPCs
(3958 cells from 3 mice); and Doxo p53-tg-CPCs (16,496 cells from 3
mice). Data are mean.+-.SD. (c) .gamma.H2A.X (green; left two
panels) in nuclei of WT-CPCs and p53-tg-CPCs stained by DAPI
(blue). DDR foci are illustrated in the same nuclei following
three-dimensional reconstruction by Imaris version 5.5.2 (right two
panels). Scale bar: 5 .mu.m. (d) Number of DDR foci counted in
nuclei of WT-CPCs and p53-tg-CPCs. In each case, 24-59 .gamma.H2A.X
positive nuclei from 3 mice were analyzed. (e) Nucleoids of WT-CPCs
and p53-tg-CPCs are stained with Vista green dye (green, left
panels). Comets are apparent after Doxo (green, right panels). (f)
Quantity of damaged DNA in nuclei of WT-CPCs and p53-tg-CPCs at
baseline (Control: WT, n=62 comets from 3 mice; p53-tg, n=70 comets
from 3 mice) and after Doxo (Doxo: WT, n=76 comets from 3 mice;
p53-tg, n=61 comets from 3 mice). *p<0.05 vs. WT Ctrl;
**p<0.05 vs. Doxo WT-CPCs; ***p<0.05 vs. p53-tg Ctrl.
[0021] FIGS. 4A-4D depict the expression of p53 and p53-dependent
genes. (a) Quantity of p53 protein by automated Wes Western
blotting in WT-CPCs (WT) and p53-tg-CPCs (p53-tg) at baseline (blue
line) and after Doxo (red line). Tracings illustrate the peak level
of p53 in the four CPC classes; n=3 in all cases. (b) The
pseudo-blots show the expression of phosphorylated p53 at Ser-18
and Ser-34, and p53 and GAPDH in the four CPC classes. (c)
Quantitative data are shown as mean.+-.SD. *p<0.05 vs. WT Ctrl.
**p<0.05 vs. WT Doxo. ***p<0.05 vs. p53-tg Ctrl. (d) mRNA
level of p53 and p53 regulated genes in the CPC classes at baseline
(Ctrl) and after Doxo; n=3 in all cases. Ct values above 35 cycles
were considered not detectable. For statistics see panel B.
[0022] FIGS. 5A-5F depict that p53 favors the functional recovery
of CPCs from oxidative stress in vitro. (a) Western blotting of
p16.sup.INK4a at baseline, after Doxo-pulse and following recovery
of WT-CPCs (WT) and p53-tg-CPCs (p53-tg); n=3 in all cases. Optical
density data are mean.+-.SD. *p<0.05 vs. WT-Control. **p<0.05
vs. WT-Doxo-pulse. ***p<0.05 vs. WT-recovery. (b) p16.sup.INK4a
labeling (upper left panel, yellow) of WT-CPCs exposed to Doxo.
Nuclei are stained by DAPI (upper right panel, blue). Phalloidin
(lower left panel, white). Merge of p16.sup.INK4a, DAPI and
phalloidin (lower right panel). Scale bar, 50 .mu.m. Fraction of
p16.sup.INK4a-positive WT-CPCs and p53-tg-CPCs at baseline,
following Doxo-pulse and after recovery; n=3 in all cases. Data are
mean.+-.SD. *p<0.05 vs. WT-Control. **p<0.05 vs.
WT-Doxo-pulse. ***p<0.05 vs. WT recovery. .sup..dagger.p<0.05
vs. p53-tg control. .sup..dagger-dbl.p<0.05 vs. p53-tg
Doxo-pulse. (c) Number of DDR foci in WT-CPCs and p53-tg-CPCs at
baseline, after Doxo-pulse and following recovery; n=3 in all
cases. For statistics see panel B. (d) Nucleoids in WT-CPCs and
p53-tg-CPCs at baseline, following Doxo-pulse and after recovery
are stained with Vista green dye (green). Comets are apparent in
Doxo-pulse and after recovery of WT-CPCs, while intact DNA is noted
in p53-tg-CPCs after recovery. (e) Damaged DNA in nuclei of WT-CPCs
and p53-tg-CPCs at baseline, after Doxo-pulse and following
recovery; n=3 in all cases. For statistics see panel B. (f)
Fraction of Ki67-positive WT-CPCs and p53-tg-CPCs following 24, 48
and 72 h recovery period; n=3 in all cases. *p<0.05 vs. 24 h.
**p<0.05 vs. 48 h.
[0023] FIGS. 6A-6B depict that p53-tg-CPCs engraft in the diabetic
heart. (FIGS. 6A-6B) Areas of myocardial damage (*) in the LV wall;
EGFP-positive (green) p53-tg-CPCs are engrafted in the majority of
these foci of injury. Cardiomyocytes are labeled by
.alpha.-sarcomeric actin (.alpha.-SA; red).
[0024] FIGS. 7A-7E depict that p53 expands the engraftment of CPCs
within the diabetic myocardium. (FIGS. 7A-7D) Areas of myocardial
regeneration shown at different magnification contain small
developing cardiomyocytes, which express EGFP and .alpha.-SA
(yellow; arrows). (FIG. 7E) Number of EGFP-positive cells per 10
mm.sup.2 of myocardium in diabetic hearts injected with WT-CPCs
(n=4) or p53-tg-CPCs (n=4). Data are mean.+-.SD. *p<0.05 vs.
WT-CPCs.
[0025] FIGS. 8A-8C depict the early commitment of p53-tg-CPCs.
(FIGS. 8A-8C) GATA4 is expressed (left, white) in EGFP-positive
cells (right, green) distributed within the damaged diabetic
myocardium. Cardiomyocytes are labeled by troponin I (right, TnI:
red).
[0026] FIGS. 9A-9D depict the expression of p53 and p53 target
genes. (a-d) Expression of Bcl2 (FIG. 9A), Bax (FIG. 9B), Aogen
(FIG. 9C) and AT1R (FIG. 9D) in cardiomyocytes of WT (n=4-5) and
p53-tg (n=5-7). Loading conditions were established by Ponceau red,
which was employed for normalization of protein expression. A
non-specific band is located above 26 kDa in the Bcl2 blot.
[0027] FIG. 10 depicts the expression of p53 and p53-dependent
genes. Time-dependent changes in the expression of p53 and
p53-related genes in p53-tg-CPCs (green line) and WT-CPCs (red
line) following exposure to Doxo; n=3 in all cases.
[0028] FIGS. 11A-1D depict CPCs and the diabetic heart. (FIGS.
11A-11D) Areas of myocardial damage (*) in the LV wall;
EGFP-positive (green) WT-CPCs are engrafted in some of these foci
of injury.
[0029] FIGS. 12A-12B depict the early commitment of WT-CPCs. (FIGS.
12A-12B) GATA4 is expressed (left, white) in EGFP-positive cells
(right, green) distributed within the damaged diabetic myocardium.
Cardiomyocytes are labeled by troponin I (right, TnI: red).
[0030] FIGS. 13A-13C depict p53 and p53-dependent genes and their
function. DNA damage activates pathways resulting in the inhibition
of cell growth and apoptosis, or DNA repair and proliferation. Red
arrows, WT; green arrows, p53-tg.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The invention described herein is based on the discovery
that cardiac progenitor cells (CPCs) with enhanced expression of
tumor suppressor p53 are useful for therapeutic purposes. Ongoing
clinical trials with autologous cardiac stem cells (CSCs) are faced
with a critical problem which is related to the modest amount of
retained cells within the damaged myocardium. Provided herein is a
strategy that overcomes in part this problem by enhancing the
number of CSCs able to engraft within the pathologic organ.
Additionally, these genetically modified CSCs can be generated in
large number, raising the possibility that multiple temporally
distinct deliveries of cells can be introduced to restore the
structural and functional integrity of the decompensated heart.
[0032] p53 is an important modulator of stem cell fate, but its
role in cardiac progenitor cells (CPCs) was unknown. An amino acid
sequence of human p53 may be found at GenBank.TM. Accession No.
BAC16799.1. The effects of a single extra-copy of p53 on the
function of CPCs in the presence of oxidative stress mediated by
doxorubicin in vitro and type-1 diabetes in vivo were tested. CPCs
were obtained from super-p53 transgenic mice (p53-tg), in which the
additional allele is regulated in a manner similar to the
endogenous protein. Old CPCs with increased p53 dosage showed a
superior ability to sustain oxidative stress, repair DNA damage and
restore cell division. With doxorubicin, a larger fraction of CPCs
carrying an extra-copy of the p53 allele recruited .gamma.H2A.X
reestablishing DNA integrity. Enhanced p53 expression resulted in a
superior tolerance to oxidative stress in vivo by providing CPCs
with defense mechanisms necessary to survive in the milieu of the
diabetic heart; they engrafted in regions of tissue injury and in
three days acquired the cardiomyocyte phenotype. This genetic
strategy of increased dosage of p53 in CPCs can be translated to
humans to increase cellular engraftment and growth, critical
determinants of successful cell therapy for the failing heart.
[0033] The tumor suppressor p53 is a major regulator of DNA repair
and cell division, cellular aging and apoptosis (Riley et al.,
2008). Phosphorylation of the N-terminal of p53 promotes DNA
repair, a process that is intimately linked to the progression of
the cell cycle. DNA repair may be less effective in old CPCs,
resulting in the accumulation of DNA lesions, a phenomenon that
favors cellular senescence. The expression of p53 increases with
aging and heart failure (Leri et al., 2003, Cheng et al., 2013) but
its actual role in CPCs is unknown; p53 may trigger apoptosis of
old cells and may induce DNA repair in cells with a younger
phenotype (Matheu et al., 2007).
[0034] Whether this potential youth promoting effect of p53 is
determined by a successful DNA damage response (DDR), mediated by
transient reparable DNA lesions in the telomeric and non-telomeric
regions of the genome, has not been defined. A prolonged DDR
signaling may result in the accumulation of non-reparable DNA foci
and initiation of cell death (Fumagalli et al., 2012). Moreover,
these intrinsic variables of CPCs have implications in the outcome
of cell therapy for the damaged heart, where the unfavorable
conditions of the recipient myocardium with high levels of
oxidative stress affect the survival and growth of the delivered
cells. These questions were addressed herein by evaluating CPC
aging in mice with enhanced expression of p53 and then by assessing
CPC engraftment in the diabetic heart that is characterized by an
environment in which the generation of reactive oxygen and
inflammation condition its evolution (Rota et al., 2006).
[0035] The super-p53 mouse (p53-tg) (Garcia-Cao et al., 2002),
which is based on a C57BL/6J genetic background, carries a single
extra gene-dose of p53. This single-copy transgene is regulated in
a manner similar to its endogenous counterpart; p53 is not
constitutively active, but undergoes post-translational
modifications in response to stress stimuli, resulting in a
moderately higher p53 activity (Garcia-Cao et al., 2006). The
increased gene dosage of p53 triggers an amplified DDR in
lymphocytes, splenocytes, embryonic fibroblasts, and epithelial
cells of the skin and intestine (Garcia-Cao et al., 2002), but its
impact on CPC aging and growth reserve has never been determined
previously. Because of these characteristics, this animal model was
considered relevant for understanding the role of p53 in CPC
function with aging and oxidative stress.
[0036] In some embodiments, the invention provides a recombinant
CPC (or a plurality of CPCs) comprising one or more copies of a
tumor suppressor p53 gene in addition to the endogenous copy of a
p53 gene. In some embodiments, a recombinant CPC comprises one, two
or three copies of a tumor suppressor p53 gene in addition to the
endogenous copy of a p53 gene. In some embodiments, recombinant
CPCs of the invention express an increased amount of p53 protein or
p53 mRNA compared to the amount expressed by an equivalent number
of CPCs (also referred to as wild-type (WT) CPCs) that do not
comprise one or more copies of a p53 gene in addition to the
endogenous copy of a p53 gene. Amounts of p53 protein or p53 mRNA
may be measured by standard assays known in the art. For example,
western blot, ELISA, northern blot or quantitative PCR may be used.
In some embodiments, recombinant CPCs of the invention express at
least about 10%, 20%, 300%, 40%, 50%, 60%, 70%, 80%, 90% or 100%
more p53 protein or p53 mRNA compared to the amount expressed by an
equivalent number of WT CPCs. In some embodiments, recombinant CPCs
of the invention express at least about 2-fold, 3-fold, 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold more p53 protein
or p53 mRNA compared to the amount expressed by an equivalent
number of WT CPCs. In some embodiments, recombinant CPCs of the
invention have enhanced expression of tumor suppressor p53.
[0037] In some embodiments, the recombinant CPCs comprising one,
two or three copies of a tumor suppressor p53 gene in addition to
the endogenous copy of a p53 gene have an improved ability to
tolerate oxidative stress compared to WT CPCs. In some embodiments,
the recombinant CPCs of the invention have restored DNA integrity
compared to WT CPCs. In some embodiments, the recombinant CPCs of
the invention have an improved proliferative capacity compared to
WT CPCs.
[0038] In one embodiment, the invention provides a pharmaceutical
composition comprising a therapeutically effective amount of
isolated cardiac progenitor cells (CPCs) and a pharmaceutically
acceptable carrier for repairing and/or regenerating damaged tissue
of a heart, wherein said isolated CPCs comprise one or more copies
of a tumor suppressor p53 gene in addition to the endogenous copy
of a p53 gene.
[0039] In another embodiment, the invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of isolated cardiac progenitor cells (CPCs) and a
pharmaceutically acceptable carrier for promoting cellular
engraftment and growth of the CPCs in damaged tissue of a heart,
wherein said isolated CPCs comprise one or more copies of a tumor
suppressor p53 gene in addition to the endogenous copy of a p53
gene.
[0040] In one embodiment, the invention provides a pharmaceutical
composition comprising a therapeutically effective amount of cells
and a pharmaceutically acceptable carrier for promoting cellular
engraftment and growth of the cells in a damaged organ or tissue,
wherein said cells comprise one or more copies of a tumor
suppressor p53 gene in addition to the endogenous copy of a p53
gene.
[0041] When recombinant CPCs comprising one, two or three copies of
a tumor suppressor p53 gene in addition to the endogenous copy of a
p53 gene are placed into a mouse with a damaged heart, long-term
engraftment of the administered CPCs occurs, and these CPCs
differentiate into, for example, cardiomyocytes, which can lead to
subsequent heart tissue regeneration and repair. The mouse
experiments indicate that isolated recombinant CPCs described
herein can be used for heart tissue regeneration in human patients
(e.g., diabetic human patients). Accordingly, provided herein are
methods for the treatment and/or prevention of a heart disease or
disorder in a subject in need thereof. In some embodiments,
provided herein is a method of treating or preventing a heart
disease or disorder in a subject in need thereof, comprising
administering isolated cardiac progenitor cells (CPCs) to the
subject, wherein the CPCs comprise one or more copies of a tumor
suppressor p53 gene in addition to the endogenous copy of a p53
gene.
[0042] In some embodiments, a subject treated by the methods and
compositions described herein has a heart disease or disorder. As
used herein, the term "heart disease or disorder", "heart disease",
"heart condition" and "heart disorder" are used interchangeably.
Heart diseases and/or conditions can include heart failure,
diabetic heart disease, rheumatic heart disease, hypertensive heart
disease, ischemic heart disease, cerebrovascular heart disease,
inflammatory heart disease and/or congenital heart disease. The
methods described herein can be used to treat, ameliorate the
symptoms, prevent and/or slow the progression of a number of heart
diseases or disorders or their symptoms. In some embodiments of all
aspects of the therapeutic methods described herein, a subject
having a heart disease or disorder is first selected prior to
administration of the recombinant CPCs.
[0043] In some embodiments, recombinant CPCs comprising one, two or
three copies of a tumor suppressor p53 gene in addition to the
endogenous copy of a p53 gene can repair damaged heart tissue in
diabetic mice. Examples of mouse models of diabetes and methods of
implanting stem cells in such mice are described in e.g., Hua et
al., PLoS One, 2014 Jul. 10; 9(7):e102198. In some embodiments,
provided herein is a method of treating or preventing a heart
disease or disorder in a diabetic subject in need thereof,
comprising administering isolated cardiac progenitor cells (CPCs)
to the subject, wherein the CPCs comprise one or more copies of a
tumor suppressor p53 gene in addition to the endogenous copy of a
p53 gene. In some embodiments, a subject treated by the methods or
compositions described herein has type 1 diabetes or type 2
diabetes.
[0044] In one embodiment, the invention provides a method of
repairing and/or regenerating damaged tissue of a heart in a
subject in need thereof comprising: (a) extracting cardiac
progenitor cells (CPCs) from a heart; (b) introducing one or more
tumor suppressor p53 genes into the CPCs of step (a); (c) culturing
and expanding said CPCs from step (b); and (d) administering a dose
of said CPCs from step (c) to an area of damaged tissue in the
subject effective to repair and/or regenerate the damaged tissue of
the heart.
[0045] In one embodiment, the invention provides a method of
promoting cellular engraftment and growth of cardiac progenitor
cells (CPCs) in damaged tissue of a heart in a subject in need
thereof comprising: (a) extracting cardiac progenitor cells (CPCs)
from a heart; (b) introducing one or more tumor suppressor p53
genes into the CPCs of step (a); (c) culturing and expanding said
CPCs from step (b); and (d) administering a dose of said CPCs from
step (c) to an area of damaged tissue in the subject effective to
promote cellular engraftment and growth of the CPCs in the damaged
tissue of the heart in a subject in need thereof.
[0046] The terms "subject", "patient" and "individual" are used
interchangeably herein, and refer to an animal, for example, a
human from whom cells for use in the methods described herein can
be obtained (i.e., donor subject) and/or to whom treatment,
including prophylactic treatment, with the cells as described
herein, is provided, i.e., recipient subject. For treatment of
those conditions or disease states that are specific for a specific
animal such as a human subject, the term subject refers to that
specific animal. The "non-human animals" and "non-human mammals" as
used interchangeably herein, includes mammals such as rats, mice,
rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The
term "subject" also encompasses any vertebrate including but not
limited to mammals, reptiles, amphibians and fish. However,
advantageously, the subject is a mammal such as a human, or other
mammals such as a domesticated mammal, e.g., dog, cat, horse, and
the like, or food production mammal, e.g., cow, sheep, pig, and the
like.
[0047] Accordingly, in some embodiments of the therapeutic methods
described herein, a subject is a recipient subject, i.e., a subject
to whom the recombinant CPCs described herein are being
administered, or a donor subject, i.e., a subject (e.g., a mouse)
from whom a heart tissue sample comprising recombinant CPCs
described herein is being obtained. A recipient or donor subject
can be of any age. In some embodiments, the subject is a "young
subject," defined herein as a subject less than 10 years of age. In
other embodiments, the subject is an "infant subject," defined
herein as a subject is less than 2 years of age. In some
embodiments, the subject is a "newborn subject," defined herein as
a subject less than 28 days of age. In one embodiment, the subject
is a human adult.
[0048] The isolated recombinant CPCs described herein can be
administered to a selected subject having any heart disease or
disorder or predisposed to developing a heart disease or disorder.
The administration can be by any appropriate route which results in
an effective treatment in the subject. In some aspects of these
methods, a therapeutically effective amount of isolated recombinant
CPCs described herein is administered through vessels, directly to
the tissue, or a combination thereof. Some of these methods involve
administering to a subject a therapeutically effective amount of
isolated recombinant CPCs described herein by injection, by a
catheter system, or a combination thereof.
[0049] As used herein, the terms "administering," "introducing",
"transplanting" and "implanting" are used interchangeably in the
context of the placement of cells, e.g., recombinant CPCs of the
invention into a subject, by a method or route which results in at
least partial localization of the introduced cells at a desired
site, such as a site of injury or repair, such that a desired
effect(s) is produced. The cells, e.g., recombinant CPCs, or their
differentiated progeny (e.g., cardiomyocytes) can be implanted
directly to the heart, or alternatively be administered by any
appropriate route which results in delivery to a desired location
in the subject where at least a portion of the implanted cells or
components of the cells remain viable. The period of viability of
the cells after administration to a subject can be as short as a
few hours, e.g., twenty-four hours, to a few days, to as long as
several years, i.e., long-term engraftment. For example, in some
embodiments of all aspects of the therapeutic methods described
herein, an effective amount of a population of isolated recombinant
CPCs is administered directly to the heart of an individual
suffering from heart disease by direct injection. In other
embodiments of all aspects of the therapeutic methods described
herein, the population of isolated recombinant CPCs is administered
via an indirect systemic route of administration, such as a
catheter-mediated route.
[0050] One embodiment of the invention includes use of a
catheter-based approach to deliver the injection. The use of a
catheter precludes more invasive methods of delivery such as
surgically opening the body to access the heart. As one skilled in
the art is aware, optimum time of recovery would be allowed by the
more minimally invasive procedure, which as outlined here, includes
a catheter approach. When provided prophylactically, the isolated
recombinant CPCs can be administered to a subject in advance of any
symptom of a heart disease or disorder. Accordingly, the
prophylactic administration of an isolated recombinant CPCs
population serves to prevent a heart disease or disorder, or
further progress of heart diseases or disorders as disclosed
herein.
[0051] When provided therapeutically, isolated recombinant CPCs are
provided at (or after) the onset of a symptom or indication of a
heart disease or disorder, or for example, upon the onset of
diabetes.
[0052] As used herein, the terms "treat," "treatment," "treating,"
or "amelioration" refer to therapeutic treatment, wherein the
object is to reverse, alleviate, ameliorate, decrease, inhibit, or
slow down the progression or severity of a condition associated
with a disease or disorder. The term "treating" includes reducing
or alleviating at least one adverse effect or symptom of a
condition, disease or disorder associated with a heart disease).
Treatment is generally "effective" if one or more symptoms or
clinical markers are reduced as that term is defined herein.
Alternatively, treatment is "effective" if the progression of a
disease is reduced or halted. That is, "treatment" includes not
just the improvement of symptoms or markers, but also a cessation
or at least slowing of progress or worsening of symptoms that would
be expected in absence of treatment. Beneficial or desired clinical
results include, but are not limited to, alleviation of one or more
symptom(s), diminishment of extent of disease, stabilized (i.e.,
not worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. In some embodiments, "treatment" and "treating" can
also mean prolonging survival of a subject as compared to expected
survival if the subject did not receive treatment.
[0053] As used herein, the term "prevention" refers to prophylactic
or preventative measures wherein the object is to prevent or delay
the onset of a disease or disorder, or delay the onset of symptoms
associated with a disease or disorder. In some embodiments,
"prevention" refers to slowing down the progression or severity of
a condition or the deterioration of cardiac function associated
with a heart disease or disorder.
[0054] In another embodiment, "treatment" of a heart disease or
disorder also includes providing relief from the symptoms or
side-effects of the disease (including palliative treatment). In
some embodiments of the aspects described herein, the symptoms or a
measured parameter of a disease or disorder are alleviated by at
least 5%, at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, or at least
90%, upon administration of a population of isolated recombinant
CPCs, as compared to a control or non-treated subject.
[0055] Measured or measurable parameters include clinically
detectable markers of disease, for example, elevated or depressed
levels of a clinical or biological marker, as well as parameters
related to a clinically accepted scale of symptoms or markers for a
disease or disorder. It will be understood, however, that the total
usage of the compositions as disclosed herein will be decided by
the attending physician within the scope of sound medical judgment.
The exact amount required will vary depending on factors such as
the type of heart disease or disorder being treated, degree of
damage, whether the goal is treatment or prevention or both, age of
the subject, the amount of cells available, etc. Thus, one of skill
in the art realizes that a treatment may improve the disease
condition, but may not be a complete cure for the disease.
[0056] In one embodiment of all aspects of the therapeutic methods
described, the term "effective amount" as used herein refers to the
amount of a population of isolated recombinant CPCs needed to
alleviate at least one or more symptoms of the heart disease or
disorder, and relates to a sufficient amount of pharmacological
composition to provide the desired effect, e.g., treat a subject
having heart disease. The term "therapeutically effective amount"
therefore refers to an amount of isolated recombinant CPCs using
the therapeutic methods as disclosed herein that is sufficient to
effect a particular effect when administered to a typical subject,
such as one who has or is at risk for heart disease.
[0057] In another embodiment of all aspects of the methods
described, an effective amount as used herein would also include an
amount sufficient to prevent or delay the development of a symptom
of the disease, alter the course of a disease symptom (for example,
but not limited to, slow the progression of a symptom of the
disease), or even reverse a symptom of the disease. The effective
amount of recombinant CPCs needed for a particular effect will vary
with each individual and will also vary with the type of heart
disease or disorder being addressed. Thus, it is not possible to
specify the exact "effective amount". However, for any given case,
an appropriate "effective amount" can be determined by one of
ordinary skill in the art using routine experimentation.
[0058] In some embodiments of all aspects of the therapeutic
methods described, the subject is first diagnosed as having a
disease or disorder affecting the heart prior to administering the
recombinant CPCs according to the methods described herein. In some
embodiments of all aspects of the therapeutic methods described,
the subject is first diagnosed as being at risk of developing a
heart disease or disorder prior to administering the recombinant
CPCs, e.g., an individual with a genetic disposition for heart
disease or diabetes or who has close relatives with heart disease
or diabetes.
[0059] For use in all aspects of the therapeutic methods described
herein, an effective amount of isolated recombinant CPCs comprises
at least 10.sup.2, at least 5.times.10.sup.2, at least 10.sup.3, at
least 5.times.10', at least 10.sup.4, at least 5.times.10.sup.4, at
least 10.sup.5, at least 2.times.10.sup.5, at least
3.times.10.sup.5, at least 4.times.10.sup.5, at least
5.times.10.sup.5, at least 6.times.10.sup.5, at least
7.times.10.sup.5, at least 8.times.10.sup.5, at least
9.times.10.sup.5, or at least 1.times.10.sup.6 recombinant CPCs or
multiples thereof per administration. In some embodiments, more
than one administration of isolated recombinant CPCs is performed
to a subject. The multiple administration of isolated recombinant
CPCs can take place over a period of time. The recombinant CPCs can
be generated from CPCs isolated from one or more donors, or from
CPCs obtained from an autologous source.
[0060] Exemplary modes of administration of recombinant CPCs and
other agents for use in the methods described herein include, but
are not limited to, injection, infusion, inhalation (including
intranasal), ingestion, and rectal administration. "Injection"
includes, without limitation, intravenous, intraarterial,
intraductal, direct injection into the tissue intraventricular,
intracardiac, transtracheal injection and infusion. The phrases
"parenteral administration" and "administered parenterally" as used
herein, refer to modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intraventricular, intracardiac,
transtracheal injection and infusion. In some embodiments,
recombinant CPCs can be administered by intravenous, intraarterial,
intraductal, or direct injection into tissue, or through injection
in the liver.
[0061] In some embodiments of all aspects of the therapeutic
methods described, an effective amount of isolated recombinant CPCs
is administered to a subject by injection. In other embodiments, an
effective amount of isolated recombinant CPCs is administered to a
subject by a catheter-mediated system. In other embodiments, an
effective amount of isolated recombinant CPCs is administered to a
subject through vessels, directly to the tissue, or a combination
thereof. In additional embodiments, an effective amount of isolated
recombinant CPCs is implanted in a patient in an encapsulating
device (see, e.g., U.S. Pat. Nos. 9,132,226 and 8,425,928, the
contents of each of which are incorporated herein by reference in
their entirety).
[0062] In some embodiments of all aspects of the therapeutic
methods described, an effective amount of isolated recombinant CPCs
is administered to a subject by systemic administration, such as
intravenous administration.
[0063] The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein refer to the administration of
population of recombinant CPCs other than directly into the heart,
such that it enters, instead, the subject's circulatory system.
[0064] In some embodiments of all aspects of the therapeutic
methods described, one or more routes of administration are used in
a subject to achieve distinct effects. For example, isolated
recombinant CPCs are administered to a subject by both direct
injection and catheter-mediated routes for treating or repairing
heart tissue. In such embodiments, different effective amounts of
the isolated recombinant CPCs can be used for each administration
route.
[0065] In some embodiments of all aspects of the therapeutic
methods described, the methods further comprise administration of
one or more therapeutic agents, such as a drug or a molecule, that
can enhance or potentiate the effects mediated by the
administration of the isolated recombinant CPCs, such as enhancing
homing or engraftment of the recombinant CPCs, increasing repair of
cardiac cells, or increasing growth and regeneration of cardiac
cells. The therapeutic agent can be a protein (such as an antibody
or antigen-binding fragment), a peptide, a polynucleotide, an
aptamer, a virus, a small molecule, a chemical compound, a cell, a
drug, etc.
[0066] As defined herein, "vascular regeneration" refers to de novo
formation of new blood vessels or the replacement of damaged blood
vessels (e.g., capillaries) after injuries or traumas, as described
herein, including but not limited to, heart disease. "Angiogenesis"
is a term that can be used interchangeably to describe such
phenomena.
[0067] In some embodiments of all aspects of the therapeutic
methods described, the methods further comprise administration of
recombinant CPCs together with growth, differentiation, and
angiogenesis agents or factors that are known in the art to
stimulate cell growth, differentiation, and angiogenesis in the
heart tissue. In some embodiments, any one of these factors can be
delivered prior to or after administering the compositions
described herein. Multiple subsequent delivery of any one of these
factors can also occur to induce and/or enhance the engraftment,
differentiation and/or angiogenesis. Suitable growth factors
include but are not limited to ephrins (e.g., ephrin A or ephrin
B), transforming growth factor-beta (TGF.beta.), vascular
endothelial growth factor (VEGF), platelet derived growth factor
(PDGF), angiopoietins, epidermal growth factor (EGF), bone
morphogenic protein (BMP), basic fibroblast growth factor (bFGF),
insulin and 3-isobutyl-1-methylxasthine (IBMX). Other examples are
described in Dijke et al., "Growth Factors for Wound Healing",
Bio/Technology, 7:793-798 (1989); Mulder G D, Haberer P A, Jeter K
F, eds. Clinicians' Pocket Guide to Chronic Wound Repair. 4th ed.
Springhouse, Pa.: Springhouse Corporation; 1998:85; Ziegler T. R,
Pierce, G. F., and Herndon, D. N., 1997, International Symposium on
Growth Factors and Wound Healing: Basic Science & Potential
Clinical Applications (Boston, 1995, Serono Symposia USA),
Publisher: Springer Verlag, and these are hereby incorporated by
reference in their entirety.
[0068] In one embodiment, the composition can include one or more
bioactive agents to induce healing or regeneration of damaged heart
tissue, such as recruiting blood vessel forming cells from the
surrounding tissues to provide connection points for the nascent
vessels. Suitable bioactive agents include, but are not limited to,
pharmaceutically active compounds, hormones, growth factors,
enzymes, DNA, RNA, siRNA, viruses, proteins, lipids, polymers,
hyaluronic acid, pro-inflammatory molecules, antibodies,
antibiotics, anti-inflammatory agents, anti-sense nucleotides and
transforming nucleic acids or combinations thereof. Other bioactive
agents can promote increased mitosis for cell growth and cell
differentiation.
[0069] A great number of growth factors and differentiation factors
are known in the art to stimulate cell growth and differentiation
of stem cells and progenitor cells. Suitable growth factors and
cytokines include any cytokines or growth factors capable of
stimulating, maintaining, and/or mobilizing progenitor cells. They
include but are not limited to stem cell factor (SCF),
granulocyte-colony stimulating factor (G-CSF),
granulocyte-macrophage stimulating factor (GM-CSF), stromal
cell-derived factor-1, steel factor, vascular endothelial growth
factor (VEGF), TGF.beta., platelet derived growth factor (PDGF),
angiopoietins (Ang), epidermal growth factor (EGF), bone
morphogenic protein (BMP), fibroblast growth factor (FGF),
hepatocyte growth factor (HGF), insulin-like growth factor (IGF-1),
interleukin (IL)-3, IL-la, IL-13, IL-6, IL-7, IL-8, IL-11, and
IL-13, colony-stimulating factors, thrombopoietin, erythropoietin,
fit3-ligand, and tumor necrosis factor .alpha.. Other examples are
described in Dijke et al., "Growth Factors for Wound Healing",
Bio/Technology, 7:793-798 (1989); Mulder G D, Haberer P A, Jeter K
F, eds. Clinicians' Pocket Guide to Chronic Wound Repair. 4th ed.
Springhouse, Pa.: Springhouse Corporation; 1998:85; Ziegler T. R.,
Pierce, G. F., and Herndon, D. N., 1997, International Symposium on
Growth Factors and Wound Healing: Basic Science & Potential
Clinical Applications (Boston, 1995, Serono Symposia USA),
Publisher: Springer Verlag.
[0070] In one embodiment of all aspects of the therapeutic methods
described, the composition described is a suspension of recombinant
CPCs in a suitable physiologic carrier solution such as saline. The
suspension can contain additional bioactive agents include, but are
not limited to, pharmaceutically active compounds, hormones, growth
factors, enzymes, DNA, RNA, siRNA, viruses, proteins, lipids,
polymers, hyaluronic acid, pro-inflammatory molecules, antibodies,
antibiotics, anti-inflammatory agents, anti-sense nucleotides and
transforming nucleic acids or combinations thereof.
[0071] In certain embodiments of all aspects of the therapeutic
methods described, the bioactive agent is a "pro-angiogenic
factor," which refers to factors that directly or indirectly
promote new blood vessel formation in the heart. The pro-angiogenic
factors include, but are not limited to ephrins (e.g., ephrin A or
ephrin B), epidermal growth factor (EGF), E-cadherin, VEGF,
angiogenin, angiopoietin-1, fibroblast growth factors: acidic
(aFGF) and basic (bFGF), fibrinogen, fibronectin, heparanase,
hepatocyte growth factor (HGF), angiopoietin, hypoxia-inducible
factor-1 (HIF-1), insulin-like growth factor-1 (IGF-1), IGF, BP-3,
platelet-derived growth factor (PDGF), VEGF-A, VEGF-C, pigment
epithelium-derived factor (PEDF), vascular permeability factor
(VPF), vitronection, leptin, trefoil peptides (TFFs), CYR61 (CCN1),
NOV (CCN3), leptin, midkine, placental growth factor
platelet-derived endothelial cell growth factor (PD-ECGF),
platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN),
progranulin, proliferin, transforming growth factor-alpha
(TGF-alpha), transforming growth factor-beta (TGF-beta), tumor
necrosis factor-alpha (TNF-alpha), c-Myc, granulocyte
colony-stimulating factor (G-CSF), stromal derived factor 1
(SDF-1), scatter factor (SF), osteopontin, stem cell factor (SCF),
matrix metalloproteinases (MMPs), thrombospondin-1 (TSP-1),
pleitrophin, proliferin, follistatin, placental growth factor
(PIGF), midkine, platelet-derived growth factor-BB (PDGF), and
fractalkine, and inflammatory cytokines and chemokines that are
inducers of angiogenesis and increased vascularity, e.g.,
interleukin-3 (IL-3), interleukin-8 (IL-8), CCL2 (MCP-1),
interleukin-8 (IL-8) and CCL5 (RANTES).
[0072] Suitable dosage of one or more therapeutic agents in the
compositions described herein can include a concentration of about
0.1 to about 500 ng/ml, about 10 to about 500 ng/ml, about 20 to
about 500 ng/ml, about 30 to about 500 ng/ml, about 50 to about 500
ng/ml, or about 80 ng/ml to about 500 ng/ml. In some embodiments,
the suitable dosage of one or more therapeutic agents is about 10,
about 25, about 45, about 60, about 75, about 100, about 125, about
150, about 175, about 200, about 225, about 250, about 275, about
300, about 325, about 350, about 375, about 400, about 425, about
450, about 475, or about 500 ng/ml. In other embodiments, suitable
dosage of one or more therapeutic agents is about 0.6, about 0.7,
about 0.8, about 0.9, about 1.0, about 1.5, or about 2.0
.mu.g/ml.
[0073] In some embodiments of all aspects of the therapeutic
methods described, the standard therapeutic agents for heart
disease are those that have been described in detail, see, e.g.,
Harrison's Principles of Internal Medicine, 15th edition, 2001, E.
Braunwald, et al., editors, McGraw-Hill, New York, N.Y., ISBN
0-07-007272-8, especially chapters 252-265 at pages 1456-1526;
Physicians Desk Reference 54th edition, 2000, pages 303-3251, ISBN
1-56363-330-2, Medical Economics Co., Inc., Montvale, N.J.
Treatment of any heart disease or disorder can be accomplished
using the treatment regimens described herein. For chronic
conditions, intermittent dosing can be used to reduce the frequency
of treatment. Intermittent dosing protocols are as described
herein.
[0074] For the clinical use of the methods described herein,
isolated populations of recombinant CPCs described herein can be
administered along with any pharmaceutically acceptable compound,
material, carrier or composition which results in an effective
treatment in the subject. Thus, a pharmaceutical formulation for
use in the methods described herein can contain an isolated
recombinant CPCs in combination with one or more pharmaceutically
acceptable ingredients.
[0075] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the therapeutic is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations, and the
like. The composition can be formulated as a suppository, with
traditional binders and carriers such as triglycerides. Oral
formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable pharmaceutical carriers are described in Remington's
Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing
Co., 1990). The formulation should suit the mode of
administration.
[0076] In one embodiment, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. Specifically, it refers to those compounds, materials,
compositions, and/or dosage forms which are, within the scope of
sound medical judgment, suitable for use in contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0077] The phrase "pharmaceutically acceptable carrier" as used
herein means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent, media (e.g., stem cell media), encapsulating material,
manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc
stearate, or steric acid), or solvent encapsulating material,
involved in maintaining the activity of, carrying, or transporting
the isolated recombinant CPCs from one organ, or portion of the
body, to another organ, or portion of the body.
[0078] Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: (1) sugars,
such as lactose, glucose and sucrose; (2) phosphate buffered
solutions; (3) pyrogen-free water; (4) isotonic saline; (5) malt;
(6) gelatin; (7) lubricating agents, such as magnesium stearate,
sodium lauryl sulfate and talc; (8) excipients, such as cocoa
butter and suppository waxes; (9) oils, such as peanut oil,
cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (10) glycols, such as propylene glycol; (11) polyols,
such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG);
(12) esters, such as ethyl oleate and ethyl laurate; (13) agar;
(14) buffering agents, such as magnesium hydroxide and aluminum
hydroxide; (15) alginic acid; (16) cellulose, and its derivatives,
such as sodium carboxymethyl cellulose, methylcellulose, ethyl
cellulose, microcrystalline cellulose and cellulose acetate; (17)
powdered tragacanth; (18) Ringer's solution; (19) ethyl alcohol;
(20) pH buffered solutions; (21) polyesters, polycarbonates and/or
polyanhydrides; (22) bulking agents, such as polypeptides and amino
acids (23) serum component, such as serum albumin, HDL and LDL;
(24) C2-C12 alcohols, such as ethanol; (25) starches, such as corn
starch and potato starch; and (26) other non-toxic compatible
substances employed in pharmaceutical formulations. Wetting agents,
coloring agents, release agents, coating agents, sweetening agents,
flavoring agents, perfuming agents, preservative and antioxidants
can also be present in the formulation. The terms such as
"excipient", "carrier", "pharmaceutically acceptable carrier" or
the like are used interchangeably herein.
[0079] In some aspects, the invention provides methods of producing
recombinant CPCs comprising one, two or three copies of a tumor
suppressor p53 gene in addition to the endogenous copy of a p53
gene.
[0080] In some embodiments, the invention provides a method of
producing a large quantity of cardiac progenitor cells (CPCs)
comprising: (a) isolating CPCs from heart tissue: (b) introducing
one or more tumor suppressor p53 genes into the CPCs of step (a);
and (c) culturing and expanding the CPCs of step (b), thereby
producing a large quantity of CPCs.
[0081] In one embodiment, the invention provides a method of
promoting cellular engraftment and growth of cells in an organ or
tissue during cell therapy, comprising: (a) extracting cells from
an organ or tissue; (b) introducing one or more tumor suppressor
p53 genes into the cells of step (a); (c) culturing and expanding
said cells from step (b); and (d) applying an amount of said cells
from step (c) to an area of damaged organ or tissue, thereby
promoting cellular engraftment and growth of cells in the damaged
organ or tissue.
[0082] In one embodiment, the invention provides a method of
producing isolated cardiac progenitor cells (CPCs) having an
improved ability to tolerate oxidative stress, comprising: (a)
isolating CPCs from heart tissue; (b) introducing one or more tumor
suppressor p53 genes into the CPCs of step (a); and (c) culturing
and expanding the CPCs of step (b), thereby producing CPCs having
an improved ability to tolerate oxidative stress compared to CPCs
from step (a).
[0083] In one embodiment, the invention provides a method of
producing isolated cardiac progenitor cells (CPCs) having restored
DNA integrity, comprising: (a) isolating CPCs from heart tissue;
(b) introducing one or more tumor suppressor p53 genes into the
CPCs of step (a); and (c) culturing and expanding the CPCs of step
(b), thereby producing CPCs having restored DNA integrity compared
to CPCs from step (a).
[0084] In one embodiment, the invention provides a method of
producing isolated cardiac progenitor cells (CPCs) having an
improved proliferative capacity, comprising: (a) isolating CPCs
from heart tissue; (b) introducing one or more tumor suppressor p53
genes into the CPCs of step (a); and (c) culturing and expanding
the CPCs of step (b), thereby producing CPCs having an improved
proliferative capacity compared to CPCs from step (a).
[0085] In some embodiments, one or more exogenous tumor suppressor
p53 genes may be introduced into CPCs isolated from a subject with
heart disease to generate recombinant CPCs. These recombinant CPCs
may then be administered to the subject from whom the parental CPCs
were isolated to treat the subject's heart disease.
[0086] The one or more exogenous tumor suppressor p53 genes may be
introduced into CPCs by any suitable methods of genetic
engineering. For example, the p53 gene may be introduced via a
viral vector, a plasmid or a nanoparticle. An exogenous p53 gene
may be operatively linked to a constitutive promoter, an inducible
promoter or a cardiac-tissue-specific promoter. In some
embodiments, an exogenous p53 gene integrates into the genome of
the recombinant CPC.
[0087] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Certain
terms employed herein, in the specification, examples and claims
are collected here.
[0088] As used herein, "in vivo" (Latin for "within the living")
refers to those methods using a whole, living organism, such as a
human subject. As used herein, "ex vivo" (Latin: out of the living)
refers to those methods that are performed outside the body of a
subject, and refers to those procedures in which an organ, cells,
or tissue are taken from a living subject for a procedure, e.g.,
isolating recombinant CPCs from heart tissue obtained from a donor
subject, and then administering the isolated recombinant CPCs to a
recipient subject. As used herein, "in vitro" refers to those
methods performed outside of a subject, such as an in vitro cell
culture experiment. For example, recombinant CPCs can be cultured
in vitro to expand or increase the number of recombinant CPCs, or
to direct differentiation of the CPCs to a specific lineage or cell
type, e.g., cardiomyocytes, prior to being used or administered
according to the methods described herein.
[0089] The term "pluripotent" as used herein refers to a cell with
the capacity, under different conditions, to commit to one or more
specific cell type lineage and differentiate to more than one
differentiated cell type of the committed lineage, and preferably
to differentiate to cell types characteristic of all three germ
cell layers. Pluripotent cells are characterized primarily by their
ability to differentiate to more than one cell type, preferably to
all three germ layers, using, for example, a nude mouse teratoma
formation assay. Pluripotency is also evidenced by the expression
of embryonic stem (ES) cell markers, although the preferred test
for pluripotency is the demonstration of the capacity to
differentiate into cells of each of the three germ layers. It
should be noted that simply culturing such cells does not, on its
own, render them pluripotent. Reprogrammed pluripotent cells (e.g.,
iPS cells) also have the characteristic of the capacity of extended
passaging without loss of growth potential, relative to primary
cell parents, which generally have capacity for only a limited
number of divisions in culture.
[0090] The term "progenitor" cell are used herein refers to cells
that have a cellular phenotype that is more primitive (i.e., is at
an earlier step along a developmental pathway or progression than
is a fully differentiated or terminally differentiated cell)
relative to a cell which it can give rise to by differentiation.
Often, progenitor cells also have significant or very high
proliferative potential. Progenitor cells can give rise to multiple
distinct differentiated cell types or to a single differentiated
cell type, depending on the developmental pathway and on the
environment in which the cells develop and differentiate.
Progenitor cells give rise to precursor cells of specific
determinate lineage, for example, certain cardiac progenitor cells
divide to give cardiac cell lineage precursor cells. These
precursor cells divide and give rise to many cells that terminally
differentiate to, for example, cardiomyocytes.
[0091] The term "precursor" cell is used herein refers to a cell
that has a cellular phenotype that is more primitive than a
terminally differentiated cell but is less primitive than a stem
cell or progenitor cell that is along its same developmental
pathway. A "precursor" cell is typically progeny cells of a
"progenitor" cell which are some of the daughters of "stem cells".
One of the daughters in a typical asymmetrical cell division
assumes the role of the stem cell.
[0092] The term "embryonic stem cell" is used to refer to the
pluripotent stem cells of the inner cell mass of the embryonic
blastocyst (see U.S. Pat. Nos. 5,843,780, 6,200,806). Such cells
can similarly be obtained from the inner cell mass of blastocysts
derived from somatic cell nuclear transfer (see, for example, U.S.
Pat. Nos. 5,945,577, 5,994,619, 6,235,970). The distinguishing
characteristics of an embryonic stem cell define an embryonic stem
cell phenotype. Accordingly, a cell has the phenotype of an
embryonic stem cell if it possesses one or more of the unique
characteristics of an embryonic stem cell such that the cell can be
distinguished from other cells. Exemplary distinguishing embryonic
stem cell characteristics include, without limitation, gene
expression profile, proliferative capacity, differentiation
capacity, karyotype, responsiveness to particular culture
conditions, and the like.
[0093] The term "adult stem cell" is used to refer to any
multipotent stem cell derived from non-embryonic tissue, including
juvenile and adult tissue. In some embodiments, adult stem cells
can be of non-fetal origin.
[0094] In the context of cell ontogeny, the adjective
"differentiated" or "differentiating" is a relative term meaning a
"differentiated cell" is a cell that has progressed further down
the developmental pathway than the cell it is being compared with.
Thus, stem cells can differentiate to lineage-restricted precursor
cells (such as a cardiac stem cell), which in turn can
differentiate into other types of precursor cells further down the
pathway (such as an exocrine or endocrine precursor), and then to
an end-stage differentiated cell, which plays a characteristic role
in a certain tissue type, and may or may not retain the capacity to
proliferate further. The term "differentiated cell" is meant any
primary cell that is not, in its native form, pluripotent as that
term is defined herein. Stated another way, the term
"differentiated cell" refers to a cell of a more specialized cell
type derived from a cell of a less specialized cell type (e.g., a
CPC) in a cellular differentiation process.
[0095] As used herein, the term "somatic cell" refers to any cell
forming the body of an organism, as opposed to germline cells. In
mammals, germline cells (also known as "gametes") are the
spermatozoa and ova which fuse during fertilization to produce a
cell called a zygote, from which the entire mammalian embryo
develops. Every other cell type in the mammalian body--apart from
the sperm and ova, the cells from which they are made (gametocytes)
and undifferentiated stem cells--is a somatic cell: internal
organs, skin, bones, blood, and connective tissue are all made up
of somatic cells. In some embodiments the somatic cell is a
"non-embryonic somatic cell", by which is meant a somatic cell that
is not present in or obtained from an embryo and does not result
from proliferation of such a cell in vitro. In some embodiments the
somatic cell is an "adult somatic cell", by which is meant a cell
that is present in or obtained from an organism other than an
embryo or a fetus or results from proliferation of such a cell in
vitro.
[0096] As used herein, the term "adult cell" refers to a cell found
throughout the body after embryonic development.
[0097] The term "phenotype" refers to one or a number of total
biological characteristics that define the cell or organism under a
particular set of environmental conditions and factors, regardless
of the actual genotype. For example, the expression of cell surface
markers in a cell. The term "cell culture medium" (also referred to
herein as a "culture medium" or "medium") as referred to herein is
a medium for culturing cells containing nutrients that maintain
cell viability and support proliferation. The cell culture medium
may contain any of the following in an appropriate combination:
salt(s), buffer(s), amino acids, glucose or other sugar(s),
antibiotics, serum or serum replacement, and other components such
as peptide growth factors, etc. Cell culture media ordinarily used
for particular cell types are known to those skilled in the
art.
[0098] The terms "renewal" or "self-renewal" or "proliferation" are
used interchangeably herein, are used to refer to the ability of
stem cells to renew themselves by dividing into the same
non-specialized cell type over long periods, and/or many months to
years.
[0099] In some instances, "proliferation" refers to the expansion
of cells by the repeated division of single cells into two
identical daughter cells.
[0100] The term "lineages" is used herein describes a cell with a
common ancestry or cells with a common developmental fate.
[0101] The term "isolated cell" as used herein refers to a cell
that has been removed from an organism in which it was originally
found or a descendant of such a cell. Optionally the cell has been
cultured in vitro, e.g., in the presence of other cells. Optionally
the cell is later introduced into a second organism or
re-introduced into the organism from which it (or the cell from
which it is descended) was isolated.
[0102] The term "isolated population" with respect to an isolated
population of cells as used herein refers to a population of cells
that has been removed and separated from a mixed or heterogeneous
population of cells. In some embodiments, an isolated population is
a substantially pure population of cells as compared to the
heterogeneous population from which the cells were isolated or
enriched from.
[0103] The term "tissue" refers to a group or layer of specialized
cells which together perform certain special functions. The term
"tissue-specific" refers to a source of cells from a specific
tissue.
[0104] The terms "decrease", "reduced", "reduction", "decrease" or
"inhibit" are all used herein generally to mean a decrease by a
statistically significant amount. However, for avoidance of doubt,
"reduced", "reduction" or "decrease" or "inhibit" typically means a
decrease by at least about 5%-10% as compared to a reference level,
for example a decrease by at least about 20%, or at least about
30%, or at least about 40%, or at least about 50%, or at least
about 60%, or at least about 70%, or at least about 80%, or at
least about 90% decrease (i.e., absent level as compared to a
reference sample), or any decrease between 10-90% as compared to a
reference level. In the context of treatment or prevention, the
reference level is a symptom level of a subject in the absence of
administering a population of recombinant CPCs.
[0105] The terms "increased", "increase" or "enhance" are all used
herein to generally mean an increase by a statically significant
amount; for the avoidance of any doubt, the terms "increased",
"increase" or "enhance" means an increase of at least 10% as
compared to a reference level, for example an increase of at least
about 20%, or at least about 30%, or at least about 40%, or at
least about 50%, or at least about 60%, or at least about 70%, or
at least about 80%, or at least about 90% increase or more, or any
increase between 10-90% as compared to a reference level, or at
least about a 2-fold, or at least about a 3-fold, or at least about
a 4-fold, or at least about a 5-fold or at least about a 10-fold
increase, or any increase between 2-fold and 10-fold or greater as
compared to a reference level. In the context of recombinant CPCs
expansion in vitro, the reference level is the initial number of
recombinant CPCs isolated from a heart sample or generated by
genetic engineering.
[0106] The term "statistically significant" or "significantly"
refers to statistical significance and generally means a two
standard deviation (2SD) below normal, or lower, concentration of
the marker. The term refers to statistical evidence that there is a
difference. It is defined as the probability of making a decision
to reject the null hypothesis when the null hypothesis is actually
true. The decision is often made using the p-value.
[0107] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0108] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0109] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
Definitions of common terms in molecular biology may be found in
Benjamin Lewin, Genes IX, published by Jones & Bartlett
Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.),
The Encyclopedia of Molecular Biology, published by Blackwell
Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8). Further, unless otherwise required by context,
singular terms shall include pluralities and plural terms shall
include the singular.
[0110] Unless otherwise stated, the present invention was performed
using standard procedures known to one skilled in the art, for
example, in Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory
Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular
Biology, Elsevier Science Publishing, Inc., New York, USA (1986);
Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et
al. ed., John Wiley and Sons, Inc.), Current Protocols in
Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and
Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S.
Bonifacino et. al. ed., John Wiley and Sons, Inc.), Culture of
Animal Cells: A Manual of Basic Technique by R Ian Freshney,
Publisher: Wiley-Liss; 5th edition (2005) and Animal Cell Culture
Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and
David Barnes editors, Academic Press, 1st edition, 1998) which are
all herein incorporated by reference in their entireties.
[0111] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0112] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about."
[0113] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited
herein, including but not limited to patents, patent applications,
articles, books, and treatises, are hereby expressly incorporated
by reference in their entirety for any purpose. In the event that
one or more of the incorporated documents or portions of documents
define a term that contradicts that term's definition in the
application, the definition that appears in this application
controls. However, mention of any reference, article, publication,
patent, patent publication, and patent application cited herein is
not, and should not be taken as an acknowledgment, or any form of
suggestion, that they constitute valid prior art or form part of
the common general knowledge in any country in the world.
[0114] In the present description, any concentration range,
percentage range, ratio range, or integer range is to be understood
to include the value of any integer within the recited range and,
when appropriate, fractions thereof (such as one tenth and one
hundredth of an integer), unless otherwise indicated. It should be
understood that the terms "a" and "an" as used herein refer to "one
or more" of the enumerated components unless otherwise indicated.
The use of the alternative (e.g., "or") should be understood to
mean either one, both, or any combination thereof of the
alternatives. As used herein, the terms "include" and "comprise"
are used synonymously.
[0115] The invention will be further clarified by the following
examples, which are intended to be purely exemplary and in no way
limiting.
EXAMPLES
Example 1: Methods
1.1. Animals
[0116] All procedures were approved by the Institutional Animal
Care and Use Committee of the Brigham and Women's Hospital. Animals
received humane care in compliance with the "Guide for the Care and
Use of Laboratory Animals" as described by the Institute of
Laboratory Animal Research Resources, Commission on Life Sciences,
National Research Council. Male and female wild-type (WT) and super
p53 transgenic (p53-tg) mice in a C57BL/6 genetic background were
studied (Garcia-Cao et al., 2002, Garcia-Cao et al., 2006). WT and
p53-tg at different ages were included in the protocols.
1.2. Ventricular Hemodynamics
[0117] Cardiac function was measured in young-adult, 3-6 months of
age, and old, 24-31 months of age, WT and p53-tg mice. Left
ventricular (LV) parameters (Leri et al., 2003, Torella et al.,
2004, Rota et al., 2007, Sanada et al., 2014) were obtained in the
closed chest preparation with a MPVS-400 system for small animals
(Millar Instruments) equipped with a PVR-1045 catheter. Under
sodium pentobarbital (50 mg/kg body weight, i.p.) anesthesia, the
right carotid artery was exposed and the pressure transducer was
inserted in the carotid artery and advanced into the LV cavity.
Data were acquired and analyzed with Chart 5 (ADInstruments)
software
1.3 Myocyte Isolation
[0118] Under pentobarbital anesthesia, the heart was excised and LV
myocytes were enzymatically dissociated (Torella et al., 2004, Rota
et al., 2007, Signore et al., 2015). Briefly, the myocardium was
perfused retrogradely through the aorta at 37.degree. C. with a
Ca.sup.2+-free solution gassed with 85% Oz and 15% N.sub.2. After 5
min, 0.1 mM CaCl.sub.2, 274 units/ml collagenase (type 2,
Worthington Biochemical Corp.) and 0.57 units/ml protease (type
XIV, Sigma) were added to the solution which contained (mM): NaCl
126, KCl 4.4, MgCl.sub.2 5, HEPES 20, Glucose 22, Taurine 20,
Creatine 5, Na Pyruvate 5 and NaH.sub.2PO.sub.4 5 (pH 7.4, adjusted
with NaOH). At completion of digestion, the LV was cut in small
pieces and re-suspended in Ca.sup.2+ 0.1 mM solution. Myocytes were
collected by differential centrifugation.
1.4 Ca.sup.2+ Transients and Sarcomere Shortening Isolated LV
myocytes were placed in a bath on the stage of an Axiovert Zeiss
Microscope and IX71 Olympus inverted microscope for the
measurements of contractility and Ca.sup.2+ transients. Experiments
were conducted at room temperature. Cells were bathed continuously
with a Tyrode solution containing (mM): NaCl 140, KCl 5.4,
MgCl.sub.2 1, HEPES 5, Glucose 5.5 and CaCl.sub.2) 1.0 (pH 7.4,
adjusted with NaOH). Measurements were performed in
field-stimulated cells by using IonOptix fluorescence and
contractility systems (IonOptix, Milton, Mass.). Contractions were
elicited by rectangular depolarizing pulses, 2 ms in duration, and
twice-diastolic threshold in intensity, by platinum electrodes
(Torella et al., 2004, Signore et al., 2015). Changes in mean
sarcomere length were computed by determining the mean frequency of
sarcomere spacing by fast Fourier transform and then frequency data
were converted to length. Ca.sup.2+ transients were measured by
epifluorescence after loading the myocytes with 10 .mu.M Fluo-3 AM
(Invitrogen). Excitation length was 480 nm with emission collected
at 535 nm using a 40.times. oil objective. Fluo-3 signals were
expressed as normalized fluorescence (F/F.sub.0).
1.5 Immunohistochemistry
[0119] Following the acquisition of the hemodynamic parameters, the
abdominal aorta was cannulated with a polyethylene catheter, PE-50,
filled with a phosphate buffer, 0.2 M, pH 7.4, and heparin, 100
U/ml. In rapid succession, the heart was arrested in diastole by
the injection of 0.15 ml of CdCl.sub.2, 100 mM, through the aortic
catheter, the thorax was opened, perfusion with phosphate buffer
was started, and the vena cava was cut to allow drainage of blood
and perfusate. After perfusion with phosphate buffer for 2 min, the
coronary vasculature was perfused for 15 min with formalin.
Subsequently, the heart was excised and embedded in paraffin (Leri
et al., 2003, Torella et al., 2004, Rota et al., 2007, Sanada et
al., 2014).
[0120] Formalin-fixed paraffin-embedded myocardial sections were
labeled with goat polyclonal anti-c-kit (R&D: cat. no. AF1356),
mouse monoclonal anti-.alpha.-sarcomeric actin (Sigma-Aldrich:
clone 5C5, cat. no. A2172) to identify CPCs and cardiomyocytes,
respectively. Nuclei were stained by DAPI. Cycling CPCs and
cardiomyocytes were recognized by labeling with mouse monoclonal
anti-Ki67 antibody (BD Biosciences: cat. no. 550609). Apoptotic and
senescent cells were recognized by the TUNEL assay (Roche: cat. no.
11684795910) and p16.sup.INK4a localization (Cell Signaling: cat.
no. 4824), respectively (Leri et al., 2003, Torella et al., 2004,
Rota et al., 2007, Sanada et al., 2014). The number of
c-kit-positive CPCs per unit area of myocardium in the atria and LV
mid-region was determined as previously described (Torella et al.,
2004, Sanada et al., 2014).
1.6 Western Blotting of Cardiomyocytes
[0121] Protein lysates of cardiomyocytes were obtained using RIPA
buffer (Sigma) and protease inhibitors. Equivalents of 50 gig of
proteins were separated on 10-12% SDS-PAGE, transferred onto PVDF
membranes (Bio-Rad) and subjected to Western blotting with mouse
monoclonal anti-Aogen (Swant: cat. no. 138), rabbit polyclonal
anti-ATIR (Millipore: cat. no. 15552), rabbit polyclonal anti-Bax
(Cell Signaling: cat. no. 7074) and rabbit polyclonal anti-Bcl2
(Cell Signaling: cat. no. D17C4) diluted 1:500-1000 in TBST or BSA
overnight at 4.degree. C. HRP-conjugated anti-IgG were used as
secondary antibodies. Proteins were detected by chemiluminescence
(SuperSignal West Femto Maximum Sensitivity Substrate, Thermo
Scientific: cat. no. 34095) and optical density was measured.
Loading conditions were determined by Ponceau S (Sigma) staining of
the membrane after transfer. Lung and kidney were used as positive
controls for Aogen and ATIR, respectively. SVT2 and B16 melanoma
cells were employed for the recognition of the bands corresponding
to Bax and Bcl2, respectively (Leri et al., 1998, Torella et al.,
2004, Goichberg et al., 2013).
1.7 CPC Isolation and Expansion
[0122] Following myocyte isolation, the small cardiac cell pool
present in the supernatant was plated in Petri dishes and, 24 h
later, c-kit positive cells were obtained by immunomagnetic sorting
(Miltenyi Biotec.: cat. no. 130-091-224) (Beltrami et al., 2003,
D'Amario et al., 2011, D'Amario et al., 2014, Sanada et al., 2014).
Subsequently, c-kit-positive cells were cultured in F12K medium
supplemented with 10% fetal bovine serum. Immunomagnetic sorting
for c-kit was repeated every three passages to select with this
protocol the fraction of cells that retained c-kit expression. This
approach was required because mouse c-kit-positive CPCs tend to
lose this surface receptor with time in culture. When possible,
immediately sorted cells were utilized; however, assays requiring
large numbers of CPCs were conducted after cell expansion.
1.8 Population Doubling Time (PDT)
[0123] CPCs were plated at low density. The number of cells per
unit area was determined at the time of seeding and 24 h later
(D'Amario et al., 2011, D'Amario et al., 2014). PDT was computed by
linear regression of log 2 values of cell number.
1.9 Proliferation, Senescence and Apoptosis
[0124] These cellular parameters were measured in baseline
conditions, following exposure to doxorubicin (Doxo; 0.5 .mu.M) for
4 h, and 24, 48 and 72 h following removal of Doxo. Cells were
fixed in 4% paraformaldehyde and the fraction of cycling cells was
determined by immunolabeling for Ki67 (eBioscience: cat. no.
14-5698-82, RRID: AB_10854564) and confocal microscopy (D'Amario et
al., 2011, D'Amario et al., 2014, Goichberg et al., 2013). The
fraction of cells that reached replicative senescence and
irreversible growth arrest was evaluated by the expression of the
senescence-associated protein p16.sup.INK4a (Abcam: cat no.
ab16123, RRID: AB_302274) (D'Amario et al., 2011, D'Amario et al.,
2014, Goichberg et al., 2013). Apoptosis was measured in CPCs at
baseline and following exposure to Doxo with the Annexin V
detection assay (BD Pharmingen). Annexin V binds to the
phosphatidylserine exposed on the outer leaflet of the cell
membrane during apoptotic cell death. CPCs were seeded in 96
multi-well clear bottom black plates (3603, Corning); 24 h later,
the medium was removed and cells were washed with PBS. FITC-Annexin
V (556547, Pharmingen) diluted in binding buffer provided by the
manufacturer was then added to the wells for a period of 30 min.
After washing in PBS, cells were stained with DAPI. FITC
(Excitation 490 nm; Emission 525 nm) and DAPI (Excitation 358 nm;
Emission 461 nm) signals were quantified using Perkin Elmer
EnVision Multilabel Reader. Apoptosis was calculated by normalizing
the FITC signal to the number of cells represented by the DAPI
signal.
1.10 DDR Foci and Comet Assay
[0125] CPCs were stained with a mouse anti-phospho-histone H2A.X
(Ser139) (Millipore: cat. no. 05-636, RRID: AB_309864). Imaris
software spot module was employed for the recognition of the
.gamma.H2A.X-positive DDR foci and 3D rendering of the data
(Goichberg et al., 2013). The number of foci per nucleus was
counted utilizing the Imaris software.
[0126] The comet assay was performed utilizing the OxiSelect Comet
Assay Kit (Cell Biolabs: cat. no. STA-351). Cells were embedded in
agarose gel and placed on top of a microscope slide. Slides were
treated with alkaline lysis buffer to remove proteins and,
subsequently, immersed in TE buffer. Electrophoresis was performed
to induce the formation of comets. Slides were stained with Vista
green dye and analyzed by fluorescence microscopy (Lorenzo et al.,
2013). The distance between the center of the head and the center
of the tail, i.e. the tail moment length, was measured with ImageJ
using comet assay plug-in. The tail moment was then calculated by
the product of the percentage of damaged DNA and the tail moment
length.
1.11 Western Blotting of CPCs
[0127] Protein lysates of CPCs were obtained using RIPA buffer
(Sigma-Aldrich: cat. no. R0278) and protease inhibitors (Torella et
al., 2004, Goichberg et al., 2013). Equivalents of 10 .mu.g
proteins were separated on 4-20% SDS-PAGE and subjected to
traditional Western blotting. Additionally, equivalents of 1 gig
proteins were analyzed with ProteinSimple Wes automated Western
blotting system (Harris, 2015). The following antibodies were
utilized: mouse monoclonal anti-p53 (Cell Signaling), rabbit
polyclonal anti-p53 (Ser 37) (Cell Signaling Technology: cat. no.
2524, RRID: AB_331743), rabbit polyclonal anti-p53 (Ser15) (Cell
Signaling Technology: cat. no. 9286S, RRID: AB_331741) and mouse
monoclonal anti-p16.sup.INK4a (Cell Signaling Technology: cat. no.
8884S, RRID: AB_11129865). Loading conditions were determined by
GAPDH
1.12 qRT-PCR
[0128] Total RNA was extracted from CPCs with TRIzol Reagent
(Invitrogen: cat. no. 15596018) and employed for the measurement of
the quantity of transcripts of p53, Mdm2, Puma, Noxa, PIDD,
Trp53inp, p16.sup.INK4a, p21.sup.Cip1, IGF-1 and PCNA. cDNA for
mRNAs was obtained from 2 .mu.g total RNA in a 20 .mu.l reaction
using High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems: cat. no. 4368814) and 100 pmole of oligo(dT).sub.15
primer (Hosoda et al., 2009, Goichberg et al., 2013). This mixture
was incubated at 37.degree. C. for 2 h. Quantitative RT-PCR was
performed with primers designed using the Vector NTI (Invitrogen)
software or downloaded from the NIH qdepot mouse primer database
(for sequences see Supplementary Methods). StepOnePlus Real-Time
PCR system (Applied Biosystems) was employed. cDNA synthesized from
100 ng total RNA was combined with Power SYBR Green PCR Master Mix
(Applied Biosystems: cat. no. 4367659) and 0.5 .mu.M each of
forward and reverse primers. Cycling conditions were as follows:
95.degree. C. for 10 min followed by 40 cycles of amplification
(95.degree. C. denaturation for 15 s, and 60.degree. C.
annealing-extension for 1 min). The melting curve was then
obtained. To avoid the influence of genomic contamination, forward
and reverse primers for each gene were located in different exons.
Reactions with primers alone were also included as negative
controls. Quantified values were normalized against the input
determined by the housekeeping gene 12-microglobulin. Real-time PCR
products were run on 2% agarose/IX TBE gel.
[0129] qRT-PCR Primer Sequences
TABLE-US-00001 Mouse p16INK4a F: (SEQ ID NO: 1)
5'-CGTGAACATGTTGTTGAGGC-3' R: (SEQ ID NO: 2)
5'-GCAGAAGAGCTGCTACGTGA-3' Mouse Igf1 F: (SEQ ID NO: 3)
5'-TGGATGCTCTTCAGTTCGTG-3' R: (SEQ ID NO: 4)
5'-CACTCATCCACAATGCCTGT-3' Mouse H2A X F: (SEQ ID NO: 5)
5'-GGTCAGAGAGACGCTTACCG-3' R: (SEQ ID NO: 6)
5'-GTAGTTGAGTCGCTGGGGAA-3' Mouse p21 F: (SEQ ID NO: 7)
5'-CCAGGATTGGACATGGTGCC-3' R: (SEQ ID NO: 8)
5'-GTGAGGAGGAGCATGAATGGAG-3' Mouse Puma F: (SEQ ID NO: 9)
5'-CGGGCTAGACCCTCTACG-3' R: (SEQ ID NO: 10)
5'-AGCCCTCCAGAAGGCAAC-3' Mouse Noxa F: (SEQ ID NO: 11)
5'-TTCAAGTCTGCTGGCACCCG-3' R: (SEQ ID NO: 12)
5'-AACGCGCCAGTGAACCCAAC-3' Mouse p53 F: (SEQ ID NO: 13)
5'-CTAGCATTCAGGCCCTCATC-3' R: (SEQ ID NO: 14)
5'-TCCGACTGTGACTCCTCCAT-3' Mouse PCNA F: (SEQ ID NO: 15)
5'-TGGATAAAGAAGAGGAGGCG-3' R: (SEQ ID NO: 16)
5'-GGAGACAGTGGAGTGGCTTT-3' Mouse PIDD F: (SEQ ID NO: 17)
5'-AAGGTTCCGTGGAGTCTGCT-3' R: (SEQ ID NO: 18)
5'-CAGAGTGGTCAGGGTTCCAT-3' Mouse Trp53inp1 F: (SEQ ID NO: 19)
5'-CTACCTCAGCACCCGCAG-3' R: (SEQ ID NO: 20)
5'-GCCCAATATCACAGACGAGA-3' Mouse Mdm2 F: (SEQ ID NO: 21)
5'-TCTGTGAAGGAGCACAGGAA-3' R: (SEQ ID NO: 22)
5'-CTGCTCTCACTCAGCGATGT-3' Mouse b2-M F: (SEQ ID NO: 23)
5'-ATGTGAGGCGGGTGGAACG-3' R: (SEQ ID NO: 24)
5'-CTCGGTGACCCTGGTCTTTTG-3'
1.13 Diabetes and CPC Injection
[0130] C57Bl/6 female mice at 3-4 months of age were treated with
streptozotocin (STZ, Sigma) for 7 consecutive days (.about.100
mg/kg body weight per day, i.p.) (Rota et al., 2006). STZ was
dissolved in 0.9/o saline solution containing 20 mM/1 sodium
citrate tribasic dehydrate (Sigma). Final STZ concentration was 5
mg/I. Animals developed hyperglycemia .about.2 weeks after the last
injection of STZ. TRUEtrack meter (Home Diagnostics, Inc.) and test
strips were employed to measure blood glucose. Animals with blood
glucose level >400 mg/dl were included in the study.
[0131] Three-four weeks after the onset of hyperglycemia, 200,000
CPCs were injected within the myocardium (4 injections of 5 .mu.l
each). Mice were sacrificed 3 days following cell transplantation.
Hearts were perfused with formalin and embedded in paraffin as
described above. Tissue sections obtained from the mid-portion of
the LV were stained for GFP (rabbit polyclonal anti-GFP, Molecular
Probes: cat. no. A-11122, RRID: AB_221569; chicken polyclonal
anti-GFP, Abcam: ab13970, RRID: AB_300798), .alpha.-sarcomeric
actin (mouse anti-.alpha.-sarcomeric actin IgM, Sigma-Aldrich: cat.
no. A2172, RRID: AB_476695), GATA4 (rabbit polyclonal anti-GATA4,
Abcam: cat. no. ab84593, RRID: AB_10670538) and troponin I (mouse
monoclonal anti-troponin I, Abcam: cat. no. ab10231, RRID:
AB_296967). The number of GFP-positive cells per 10 mm.sup.2 of
myocardium was measured throughout the entire cross-section of the
LV.
1.14 Data Analysis
[0132] Data are presented as mean.+-.SD. The Shapiro-Wilk test was
utilized to define the normality of value distribution. In case of
normal distribution, significance between two groups was determined
by unpaired two-tailed Student's t-test. For multiple comparisons,
the ANOVA test with the Bonferroni parametric correction was
employed. When the normality test failed, the Mann-Whitney Rank Sum
Test and the Kruskal-Wallis One Way ANOVA were employed. In all
cases, p<0.05 was considered significant (McDonald, 2014).
Example 2: Results
[0133] 2.1 p53 does not Alter the Mechanical and Growth Properties
of Cardiomyocytes
[0134] The overexpression of p53 results in premature organism
aging and animal mortality (Serrano and Blasco, 2007). The shorter
lifespan may be due to defects in cardiac performance and myocyte
mechanics, commonly found in the old failing heart (Leri et al.,
2003, Torella et al., 2004, Signore et al., 2015). Therefore, we
determined whether an increase in p53 gene dosage had a negative
effect on ventricular hemodynamics and the electro-mechanical
properties of cardiomyocytes. Wild-type (WT) and p53-tg mice at 3-6
and 24-31 months of age were studied. At both ages, left
ventricular (LV) systolic pressure, LV end-diastolic pressure, LV
developed pressure, and LV+dP/dt and -dP/dt did not differ in
p53-tg and WT mice (FIG. 1A).
[0135] Moreover, Ca.sup.2+ transient amplitude, sarcomere
shortening, and the timing parameters of Ca.sup.2+ transient and
sarcomere shortening were measured in isolated LV myocytes. In all
cases, no differences were found (FIGS. 1B-1C), suggesting that the
physiological properties of the LV and cardiomyocytes were
preserved in WT mice as a function of age, and a single extra
gene-dose of p53 did not alter the function of the old heart. These
observations are consistent with previous results in which aging
effects have not been detected in WT 26 month-old C57BL/6J mice
(Sanada et al., 2014).
[0136] To define further the characteristics of cardiomyocytes, the
degree of cell replication and death was evaluated in young-adult,
8-11 months, and old, 20-25 months, WT and p53-tg mice. The
fraction of cycling Ki67-positive myocytes and the percentage of
apoptotic myocytes were similar in young WT and p53-tg and
increased equally with age in both groups of mice (FIG. 2A-2B).
However, only the increase in cell death in p53-tg hearts was
statistically significant. Moreover, the number of senescent
p16.sup.INK4a-positive cardiomyocytes was comparable in 18-33
month-old WT and p53-tg (FIG. 2C), supporting the notion that the
extra copy of p53 did not promote myocardial aging. This finding is
typical of this model in which the p53 transgene is physiologically
regulated and it is not constitutively active. Conversely,
transgenic and mutant mice with chronically active p53 signaling
are characterized by shortened lifespan (Matheu et al., 2008).
[0137] Cardiomyocyte apoptosis and aging are controlled in part by
the expression of the p53-dependent genes, Bax and Bcl2, and the
p53-regulated genes, angiotensinogen (Aogen) and angiotensin II
(Ang II) type-1 receptors (AT1R) (Leri et al., 1998, Leri et al.,
1999, Dimmeler and Leri, 2008, Xu et al., 2010). These parameters
were measured in myocytes isolated from p53-tg and WT mice at 25
months of age. At the protein level, the quantity of the
pro-apoptotic gene Bax and the anti-apoptotic gene Bcl2 was similar
in WT and p53-tg myocytes (FIG. 9). Additionally, the levels of
Aogen and ATIR did not differ in the two groups of cardiomyocytes
(FIG. 9). Thus, an extra copy of p53 has no negative effects on
cardiac performance, myocyte mechanics, Ca.sup.2+ transient, and
cardiomyocyte growth, senescence and death.
2.2 p53 Preserves a Younger CPC Phenotype
[0138] CPC niches are preferentially located in the atrial
myocardium (Sanada et al., 2014) so that a quantitative analysis
was performed in this anatomical region of WT at 24-25 months and
p53-tg at 24-31 months. The frequency of CPCs was significantly
higher in p53-tg, while the fraction of replicating Ki67-positive
CPCs was similar in the two groups (FIG. 2d, e). Because of these
two variables, a larger pool of cycling CPCs was present in the
atria of p53-tg mice.
[0139] To evaluate the growth reserve of CPCs, these cells were
isolated from the myocardium of p53-tg at 26-30 months and WT at
23-25 months; cells were expanded in vitro and population doubling
time (PDT) was determined at P10-P13. PDT was 47% shorter in
p53-tg-CPCs than in WT-CPCs (FIG. 2f). Moreover, the percentage of
Ki67-positive CPCs at P10-P13 was 3.9-fold higher in p53-tg-CPCs
(1528/4561; 33.5%) than in WT-CPCs (543/6235; 8.7%) (FIG. 2g). At
later passages, P16-P17, p16.sup.INK4a comprised 2.9% of WT-CPCs
(36/1255; 2.9%) and only 0.03% of p53-tg-CPCs (1/3275; 0.03%) (FIG.
2h). However, apoptosis was 35% higher in p53-tg-CPCs (FIG. 2i),
despite the lower number of senescent cells. Thus, an extra copy of
the p53 allele preserves a younger CPC phenotype after propagation
in vitro and prevents the accumulation of senescent CPCs by
potentiating cell death.
2.3 p53 Increases the Repair of DNA Damage in CPCs
[0140] Reactive oxygen species (ROS) induce foci of injury in the
telomeric and non-telomeric DNA; this affects the growth and
viability of the target cells (Schieber and Chandel, 2014). To
evaluate whether p53-tg-CPCs had a superior, equal or inferior
ability to sustain ROS-mediated DNA damage than WT-CPCs, these stem
cell classes were exposed to a low dose of doxorubicin (Doxo) which
is coupled with the formation of DNA strand breaks (Goichberg et
al., 2013).
[0141] The .gamma.H2A.X protein accumulates at regions of DNA
strand breaks, allowing the recognition of DNA damage (Mohrin et
al., 2010, Goichberg et al., 2013). The localization of
.gamma.H2A.X increased from 4.7% (200/4284; 4.7%) to 29%
(1148/3958; 29%) in WT-CPCs and from 2.2% (296/13334; 2.2%) to
73.8% (12,185/16496; 73.8%) in p53-tg-CPCs (FIGS. 3A-3B). These
results suggest that p53-tg-CPCs were 2.5-fold more efficient than
WT-CPCs in recruiting .gamma.H2A.X at the sites of DNA damage, a
process necessary for the initiation of DNA repair (Fumagalli et
al., 2012). However, the enhanced recruitment of .gamma.H2A.X at
the sites of DNA damage in p53-tg-CPCs may be independent from the
extra copy of the p53 allele; p53-tg-CPCs possess a younger cell
phenotype (see FIGS. 2H-2I), which may determine the higher
efficiency of DNA repair in this progenitor cell class.
[0142] DDR foci correspond to the localization of the .gamma.H2A.X
protein at the level of DNA lesions. In the presence of Doxo, the
incidence of DDR foci per nucleus (p53-tgCPCs, baseline: 6.3;
WT-CPCs, baseline: 5.1; p53-tgCPCs, Doxo: 79; WT-CPCs, Doxo: 63)
increased markedly and in a similar manner, 12-fold, in p53-tg-CPCs
and WT-CPCs (FIGS. 3C-3D), although a larger fraction of
p53-tg-CPCs recruited .gamma.H2A.X, as shown in FIG. 3B. High
values of DDR foci per nucleus may indicate an effective completion
of DNA repair and/or a more extensive DNA damage (Lukas et al.,
2011). To test this possibility the degree of DNA damage in the two
categories of CPCs was determined by the Comet assay (Lorenzo et
al., 2013).
[0143] CPCs were embedded in agarose on microscope slides and lysed
to form nucleoids. Electrophoresis was performed to identify
structures resembling comets at fluorescence microscopy (FIG. 3E).
The fluorescence intensity of the tail (damaged DNA) relative to
the head (intact DNA) reflects the percentage of DNA damage; 61-76
comets were analyzed in WT-CPCs and p53-tg-CPCs in the absence and
presence of Doxo. The distance between the center of the head and
the center of the tail, i.e. the tail moment length, indicates the
frequency of DNA strand breaks. The tail moment was calculated by
the product of the percentage of damaged DNA and the tail moment
length. The tail moment provides a parameter that comprises both
the extent of DNA damage and the frequency of DNA strand breaks;
this index was found to be comparable at baseline and to increase
similarly in p53-tg-CPCs and WT-CPCs following Doxo (FIG. 3F).
Thus, the extent of damaged DNA promoted by oxidative stress was
analogous in the two CPC classes (see FIG. 3D), but a larger
fraction of cells carrying an extra copy of the p53 allele
recruited .gamma.H2A.X (see FIG. 3B), possibly enhancing DNA
repair.
2.4 p53 Enhances the Expression of Genes Regulating DDR
[0144] The tumor suppressor p53 trans-activates several genes
implicated in the cell cycle and apoptosis (Riley et al., 2008),
and an increase in p53 gene dosage may impact on the function of
CPCs. Therefore, the expression of p53 and its target genes was
measured in p53-tg-CPCs and WT-CPCs in the absence and presence of
Doxo. At baseline, the quantity of p53 was similar in the two stem
cell categories (FIGS. 4A-4C). After 4 h of Doxo, p53 levels
increased and p53 phosphorylation at Ser-18, a post-translational
modification required for p53 DNA binding, was present in both
WT-CPCs and p53-tg-CPCs. At baseline, p53 phosphorylation at Ser-34
was high in WT-CPCs and in p53-tg-CPCs and with Doxo decreased in
both stem cell categories (FIGS. 4B-4C). Together with other sites
of post-translational modifications, phosphorylation of p53 at
Ser-34 is relevant to DDR (Loughery and Meek, 2013).
[0145] The expression of p53 and other genes (Riley et al., 2008)
implicated in inhibition of p53 activity (Mdm2), induction of
apoptosis (Puma and Noxa), protection from oxidative stress
(Trp53inp), cellular senescence (p16.sup.INK4a), cell cycle arrest
and DNA repair (p21.sup.Cip1), and proliferation (IGF-1 and PCNA),
was measured by qRT-PCR. The expression of PIDD was also
determined; PIDD is a master regulator of cell fate decision,
playing a critical role in DNA repair, cell proliferation, survival
and death (Bock et al., 2012).
[0146] At baseline, p53, PIDD, IGF-1 and PCNA transcripts were
higher and p21.sup.Cip1 was lower in p53-tg-CPCs than in WT-CPCs,
possibly reflecting the enhanced proliferative activity of cells
with an extra copy of the p53 gene (FIG. 4D). With Doxo treatment,
Mdm2, Puma and p21.sup.Cip1 increased mostly in p53-tg-CPCs,
suggesting that p21.sup.Cip1 promoted cell cycle arrest and favored
DNA repair. However, p16.sup.INK4a was decreased in p53-tg-CPCs.
PIDD and Trp53inp were upregulated in p53-tg-CPCs and WT-CPCs, but
the changes in Trp53inp were greater in p53-tg-CPCs; thus, the
protection from oxidative stress was enhanced in p53-tg-CPCs (FIG.
4D). With Doxo, the expression of IGF-1 and PCNA decreased in
p53-tg-CPCs and these changes are consistent with activation of the
DNA repair process. In WT-CPCs, Doxo led to an attenuation of IGF-1
and an upregulation of Noxa, which may mediate cell apoptosis.
[0147] The temporal changes in the expression of p53, Mdm2,
p21.sup.Cip1, Noxa, PIDD, Trp53inp and Puma were evaluated in
p53-tg-CPCs and WT-CPCs from time 0 to 120 min following
Doxo-treatment (FIG. 10). In p53-tg-CPCs, the expression of p53
appeared to increase earlier than the upregulation of Mdm2,
p21.sup.Cip1, PIDD, Trp53inp and Puma. These adaptations suggest
that oxidative stress was coupled with a rapid response in the
genes modulating p53 function, growth arrest, oxidative DNA damage
and repair, and cell death. Conversely, in WT-CPCs, the modest
increase in p53 was associated with a time-dependent increase in
the pro-apoptotic gene Noxa (FIG. 10).
[0148] The expression of Noxa and Puma is essential for
p53-mediated apoptosis; in this regard, the deletion of these two
genes prevents cell death in response to stimuli leading to
upregulation of p53 activity (Valente et al., 2013). The
differential expression of Noxa and Puma in WT-CPCs and p53-tg-CPCs
with oxidative stress may depend on the distinct post-translational
modifications of p53, which condition the transactivation of
specific target genes. Additionally, .gamma.H2A.X, which is more
effectively recruited at the sites of DNA damage in p53-tg-CPCs,
promotes upregulation of Puma independently from p53 signaling (Xu
et al., 2016). Thus, p53 is a critical determinant of stem cell
fate and an extra copy of the p53 allele positively impacts on the
survival and growth of CPCs.
2.5 p53 Promotes DNA Repair and Recovery of CPC Growth
[0149] To determine whether the distinct response of CPC classes to
oxidative stress was translated in a differential recovery in
function, p53-tg-CPCs and WT-CPCs were exposed to Doxo for 4 h
(Doxo-pulse) and, after Doxo removal, cellular senescence, DNA
repair and proliferation were measured following a 72-hour recovery
period (Recovery). After recovery, p16.sup.INK4a expression was
barely detectable by Western blotting in p53-tg-CPCs, but was
upregulated in WT-CPCs (FIG. 5A). Similarly, by immunolabeling and
confocal microscopy, a small fraction of p16.sup.INK4a-positive
cells was identified in p53-tg-CPCs, while numerous WT-CPCs
expressed p16.sup.INK4a [FIG. 5b; (WT-CPCs: control=6/610, 0.98%;
Doxo pulse=22/1742, 1.26%; Recovery=150/2877, 5.2%) (p53-tg-CPCs:
control=6/1903, 0.3.2%; Doxo pulse=3/4293, 0.07%; Recovery=32/3473,
0.9%)]. Importantly, following recovery, the number of DDR foci and
the tail moment decreased dramatically in p53-tg-CPCs; however,
these parameters remained high in WT-CPCs (FIGS. 5C-5E).
Additionally, a progressive increase in cell proliferation was
observed in p53-tg-CPCs from 24 to 48 and 72 h after the removal of
Doxo [FIG. 5F; (WT-CPCs: 24 h=143/8167, 1.7%; 48 h=511/8405, 6.1%;
72 h=270/4915, 5.4%) (p53-tg-CPCs: 24 h=305/7902, 0.3.9%; 48
h=1443/13635, 11%; 72 h=1032/6246, 17%)]. In contrast, the
reinstitution of cell proliferation was modest in WT-CPCs. Thus,
following oxidative stress, an extra copy of the p53 allele
potentiates the ability of CPCs to reestablish the integrity of the
DNA, leading to a relevant restoration of cell growth.
2.6 p53 Increases the Engraftment of CPCs in the Diabetic Heart
[0150] The in vitro results discussed thus far have suggested that
p53-tg-CPCs have the capacity to grow extensively in vitro and are
more resistant to ROS than WT-CPCs. These two characteristics are
critical for the successful implementation of cell therapy for the
pathologic heart. Tissue reconstitution involves isolation, in
vitro expansion and delivery of CPCs to the damaged myocardium,
where the hostile environment and high levels of oxidative stress
(Kizil et al., 2015) interfere with the cardiac repair process and
cardiomyocyte regeneration (Broughton and Sussman, 2016). To test
whether p53-tg-CPCs retained in vivo the properties documented in
vitro, both WT-CPCs and p53-tg-CPCs were injected intramyocardially
in diabetic mice 3-4 weeks after the administration of
streptozotocin when the blood glucose level was >400 mg/dl. This
model was selected because is characterized by enhanced oxygen
toxicity (Rota et al., 2006). Animals, 4 in each group, were
sacrificed 3 days later when engraftment of CPCs is expected to be
completed and cell differentiation may begin to occur. This
protocol was based on previous observations concerning the
engraftment and lineage specification of c-kit-positive
hematopoietic stem cells delivered to the damaged myocardium (Rota
et al., 2007). Four injections of EGFP-labeled CPCs were performed
in different sites of the LV wall. Diabetes was characterized by
foci of tissue injury where both WT-CPCs and p53-tg-CPCs homed
(FIG. 6; FIG. 11) and began to acquire the cardiomyocyte phenotype
(FIGS. 7A-7D). Quantitatively, the number of EGFP-positive cells in
the LV myocardium was 2350/10 mm.sup.2 and 1590/10 mm.sup.2 in
diabetic mice treated with p53-tg-CPCs and WT-CPCs, respectively
(FIG. 7E).
[0151] Additionally, clusters of EGFP-positive cells in the early
stage of myocyte commitment were recognized by the expression of
the transcription factor GATA4 (FIG. 8; FIG. 12). The volume of
these developing myocytes can be expected to increase with time and
reach in part an adult cell phenotype, as observed previously by in
situ activation of endogenous CPCs after myocardial infarction.
Importantly, the generation of parenchymal cells in that setting
was associated with growth of both resistance arterioles and
capillary profiles (Urbanek et al., 2005). Thus, CPCs carrying an
extra copy of the p53 gene have an intrinsic advantage and a
superior cellular regenerative response after injection in the
diabetic heart.
Example 3: Discussion
[0152] The results of the current study indicate that CPCs obtained
from the heart of old mice carrying an extra gene-dose of p53 can
be propagated extensively in vitro retaining an impressive growth
reserve at late passages. Based on this genetic modification, large
quantities of CPCs can be generated, raising the possibility that
multiple temporally distinct deliveries of cells can be introduced
to restore the structural and functional integrity of the damaged
myocardium. This critical aspect of autologous cell therapy has
recently been documented experimentally (Tokita et al., 2016).
Although it might be intuitively obvious that one injection of CPCs
cannot reverse cardiac pathology, this work has provided the
information needed for the development of a better strategy for the
treatment of human heart failure. Thus, a large number of the
patient's own CPCs is required, together with the ability of the
expanded cells to engraft within the unfavorable environment of the
diseased heart.
[0153] As documented in the current study, the enhanced expression
of p53 leads to a complex cellular response which involves a
network of genes implicated in DNA repair and cell proliferation,
and cellular senescence and apoptosis (FIG. 13). The extra copy of
the p53 gene improves the ability of CPCs to sustain oxidative
stress, an adaptation mediated by a rapid restoration of the
integrity of the DNA and cell division. The prompt and efficient
recruitment of DDR proteins at the sites of DNA strand breaks in
p53-tg-CPCs reflects the mechanism needed to counteract the
consequences of DNA damaging agents, typically present in the
diabetic, old and failing heart (Frustaci et al., 2000, Dimmeler
and Leri, 2008, Goichberg et al., 2014).
[0154] Conversely, CPCs with unmodified quantity of endogenous p53
are less resistant to oxidative stress and fail to mend
proficiently DNA strand breaks, a defect that results in
irreversible growth arrest and cell death. Thus, p53-tg-CPCs have a
significant biological and therapeutic advantage with respect to
WT-CPCs; they manifest a higher survival rate when delivered in
vivo enhancing cell homing and potentially myocardial regeneration.
The increased dosage of p53 provides CPCs with critical defense
mechanisms necessary for the cells to remain viable in the adverse
milieu of the diabetic and failing heart.
[0155] Despite severe hyperglycemia and its toxic consequences,
p53-tg-CPCs engraft more effectively than WT-CPCs within the sites
of damage present throughout the myocardium of diabetic mice and
initiate a reparative process. The difference in the magnitude of
cell homing observed with WT-CPCs and p53-tg-CPCs in the presence
of diabetes underscores how critical is the function of p53 in
enhancing the ability of the delivered cells to colonize the
injured ventricle, a condition necessary for the successful
replacement of cardiomyocytes lost as a result of cardiac pathology
(Leri et al., 2015).
[0156] Human CPCs have recently been introduced in the management
of heart failure in patients suffering from post-infarction
ischemic cardiomyopathy with encouraging results (Chugh et al.,
2012, Makkar et al., 2012). However, several clinical trials with a
variety of progenitor cells have been performed in the last decade
in similar patient cohorts but the outcome has been inconsistent
(Afzal et al., 2015). Despite the use of large number of cells,
there is general agreement that the fraction of engrafted cells is
miniscule and this limitation precludes an efficient recovery of
the injured myocardium. The strategy employed here may overcome in
part this problem and make stem cell therapy more effective in
restoring the structural and functional integrity of the
decompensated human heart.
[0157] Poor survival and limited retention of adoptively
transferred stem cells in the pathologic heart may reduce
significantly the efficacy of regenerative therapy. Stem cell
viability is influenced by the ischemic condition and inflammatory
response of the recipient myocardium and the intrinsic properties
of donor cells (Broughton and Sussman, 2016). Several strategies
have been utilized to reduce the susceptibility of the delivered
cells to die and prolong the window of time available for their
engraftment within the damaged myocardium. Preconditioning of CPCs
with a variety of cytokines potentiates their resistance to
oxidative stress, favoring their migration and recruitment.
[0158] A more prolonged effect is obtained when stem cells are
genetically modified to express anti-apoptotic mediators. Canonical
regulators of myocyte survival, oncogenic proteins and factors
involved in the development of embryonic-fetal myocyte progenitors
have been employed (Broughton and Sussman, 2016). The
serine/threonine Pim-1 kinase which is a downstream target of Akt
favors the engraftment and lineage commitment of CPCs and long-term
myocardial regeneration (Cottage et al., 2012, Mohsin et al.,
2013). CPCs obtained by p53-tg mice show characteristics similar to
those observed in the presence of Pim-1: the increased
proliferation and delayed cell aging in vitro are accompanied by
enhanced engraftment and survival in vivo. The extra gene copy of
p53, however, provides an additional advantage through the
selective depletion of old damaged stem cells maintaining a pool of
progenitors with a younger cell phenotype.
[0159] The structural integration of the delivered CPCs with the
recipient organ is the primary event that conditions the long-term
recovery of the lost myocardium. However, in the current study, we
did not evaluate the durability of the process, which will be
determined in future work with the expectation that the injected
p53-tg-CPCs will differentiate and generate mature,
functionally-competent cardiomyocytes, together with the required
coronary microcirculation. At the early time point, the injected
WT-CPCs and p53-tg-CPCs were restricted to the injured regions of
the ventricular wall. The microenvironment of the damaged diabetic
myocardium is unquestionably hostile although obligatory for cell
homing. The transplantation of progenitor cells in the intact
tissue results in cell apoptosis (Tillmanns et al., 2008).
[0160] The function of p53 as fate modulator has been studied in
several stem cell systems, where it exerts opposite functions,
which appear to be context and cell type dependent. p53
orchestrates the polarity of self-renewing divisions in neural stem
cells and coordinates the timing for cell fate specification
(Quadrato and Di Giovanni, 2012). During steady-state
hematopoiesis, the basal-level of p53 activity regulates the
quiescence and self-renewal of hematopoietic stem cells (HSCs)
expanding the immature cell pool (Liu et al., 2009a). This
phenomenon may overcome the decline in HSC function observed with
aging, although a larger pool of HSCs with intense self-renewal
capacity may favor the development of leukemia (Asai et al.,
2011).
[0161] The ability of the heart to maintain the steady state and
respond to injury declines with aging and diabetes (Eming et al.,
2014). The composition of the stem cell pool changes in both cases,
favoring the accumulation of cells that do not self-renew and may
manifest a skewed pattern of lineage choices. Apoptosis is
restricted to p16.sup.INK4a-positive CPCs, but the process of
clearance of old CPCs is inefficient resulting in their progressive
accumulation (Sanada et al., 2014). Enhanced p53 expression
corrects the abnormal behavior of CPCs, modifying their fate. As
shown here, in the presence of oxidative stress, p53 upregulates
the expression of Trp53inp and PIDD in CPCs ameliorating DDR
Additionally, p53 increases the level of Puma favoring apoptosis of
damaged CPCs. Thus, p53, through cell death activation, prevents
the secretory activity of senescent CPCs which release a variety of
molecules exerting pro-aging effects on the surrounding young cells
(Tchkonia et al., 2013).
[0162] Stem cells constitute a long-lived replicative cell
population that experiences prolonged periods of quiescence. Stem
cell quiescence protects from endogenous stresses mediated by cell
respiration and DNA division, but these functions are attenuated by
oxidative stress. Old, rarely dividing cells show more .gamma.H2AX
foci than actively proliferating cells (Rossi et al., 2007, Liu et
al., 2009b), since the molecular control of DNA repair is
intimately linked to the progression of the cell cycle.
Importantly, the extent of DNA damage is comparable in WT-CPCs and
p53-tg-CPCs but the enhanced expression of p53 expands the pool of
cells displaying DDR foci. This biological response supports the
view that CPCs genetically modified to express physiologically
regulated p53 are protected from environmental stimuli and genomic
lesions. DNA repair maintains genomic integrity and attenuates the
rate of aging of p53-tg-CPCs.
[0163] Whether the enhanced expression of p53 improves the
intrinsic properties of CPCs, or the intact resident stem cell
compartment is activated by the intramyocardial injection of
specific growth factors, these cells are responsible for myocyte
and coronary vessel regeneration (Beltrami et al., 2003, Sanada et
al., 2014, Liu et al., 2015). The replicative reserve of
c-kit-positive CPCs predicts the evolution of ischemic
cardiomyopathy following revascularization in humans (D'Amario et
al., 2014) and profound defects in human CPC function are present
with advanced heart failure (Urbanek et al., 2003, Urbanek et al.,
2005) and in the decompensated senescent human heart (Chimenti et
al., 2003). CPCs are the critical determinant of human cardiac
pathology and strategies increasing their growth and reparative
process may have important clinical implications.
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Sequence CWU 1
1
24120DNAMus sp. 1cgtgaacatg ttgttgaggc 20220DNAMus sp. 2gcagaagagc
tgctacgtga 20320DNAMus sp. 3tggatgctct tcagttcgtg 20420DNAMus sp.
4cactcatcca caatgcctgt 20520DNAMus sp. 5ggtcagagag acgcttaccg
20620DNAMus sp. 6gtagttgagt cgctggggaa 20720DNAMus sp. 7ccaggattgg
acatggtgcc 20822DNAMus sp. 8gtgaggagga gcatgaatgg ag 22918DNAMus
sp. 9cgggctagac cctctacg 181018DNAMus sp. 10agccctccag aaggcaac
181120DNAMus sp. 11ttcaagtctg ctggcacccg 201220DNAMus sp.
12aacgcgccag tgaacccaac 201320DNAMus sp. 13ctagcattca ggccctcatc
201420DNAMus sp. 14tccgactgtg actcctccat 201520DNAMus sp.
15tggataaaga agaggaggcg 201620DNAMus sp. 16ggagacagtg gagtggcttt
201720DNAMus sp. 17aaggttccgt ggagtctgct 201820DNAMus sp.
18cagagtggtc agggttccat 201918DNAMus sp. 19ctacctcagc acccgcag
182020DNAMus sp. 20gcccaatatc acagacgaga 202120DNAMus sp.
21tctgtgaagg agcacaggaa 202220DNAMus sp. 22ctgctctcac tcagcgatgt
202319DNAMus sp. 23atgtgaggcg ggtggaacg 192421DNAMus sp.
24ctcggtgacc ctggtctttt g 21
* * * * *